It is showed by authors that using of 8-para(toluenesulphonylamino)quinoline — a derivative of 8-oxyquinolin result histochemical revealing by using of fluorescent microscopy of Zn+2-ions in cells of tissue of prostate, in pancreatic B-cells and in salivary glands contains a large amounts of ions. This method is high sensitive and high specific for revealing of Zn+2-ions and there are only one step of staining procedures. Meanwhile it is possible to use only fresh frozen sections of tissues for investigation within short time limited by 15–20 min.
Background. Pancreatic B-cells contains a large amount of Zn+2-ions [1–3] as salivary glands and prostate. In B-cells Zn+2-ions take part in processes of biosynthesis of insulin as in storage by forming of zincinsulin complex [4, 5]. It is known that Zn+2-ions in B-cells formed with insulin a deposited form of hormone as Zn+2-insulin complex [4]. Proinsulin forms a Zn+2-ions containing hexamer soon after its synthesis. In addition the zinc ions enhance proinsulin solubility and render insulin insoluble. Pancreas of animals as of Human contain Zn+2-ions [6].
There are between insulin and zinc content in B-cells: decreasing of insulin content accompanied by decreasing of amount of Zn+2-ions and in opposite in intact B-cells a large amount of insulin accompanied by a large amount of Zn+2-ions. Meanwhile for estimate ability of B-cells for storage of insulin in cells it is necessary to use method of staining of zinc-ions.
Some diabetogenic derivatives of 8-oxyquinolin [8OXQ] possess high chemical affinity for Zn+2-ions and in vitro formed color complexes as Zn+2-chelator [7]. One of tits, a 8-para(toluenesulphonylamino)quinoline [TSQ] is used for color revealing of Zn+2-ions in solutions [8].
Aim of work: 1) to investigate Zn+2-ions content in B-cells using staining by TSQ in pancreas tissue, in prostate and salivary glands of intact animals.
Methods. Animals: 8 Rabbits 2,2–2,6 kg. Frozen sections of Pancreas tissue as of Prostate and Salivary glands were used. Group 1: A) Staining of Zn+2-ions in B-cells on sections of intact animal’s pancreas tissue, prostate and of salivary gland using of 0,4 % acetone solution of TSQ. B) Injection of TSO, 38,6 mg/kg and fluorescent microscopy of frozen section of pancreas.
Staining procedures
- Staining procedures for sections of pancreas using fluorescent reagent 8PTSQ: 0,4 % acetone solution of TSQ prepared using NH4OH 25 %-solution. Staining procedures: a few drops of 8PTSQ solution place on frozen sections for 10 sec.; 3 times washing by distilled water and investigation on UV-light microscope with measuring of intensity of fluorescence (control intensity of fluorescence of exocrine tissue’s cells was accepted for 1,00); length of wave of UV-light 360–370 For quantitative estimation of results of measuring intensity of fluorescence parameter K was calculated as rate: intensity of fluorescence of B-cells IF1/intensity of fluorescence of exocrine tissue cells IF2 (IF1/IF2);
- Preparing of TSQ solution for injection (vital staining of Zn+2-ions in islets, prostate and salivary glands): 25 mg of 8PTSQ (Institiute for High Pure Chemicals, Moscow) was dissolved in 70 % Ethanol at +700 Celsius and injected to Rabbits 36,5–38,8 mg/kg.
- Staining of insulin and Zn+2-ions content in B-cells of animals with experimental diabetes caused by injection of
Results
- Intact animals. Intensive fluorescence of complex Zn+2-ions-TSQ as positive reaction for Zn+2-ions in cytoplasm of B-cells was revealed in cytoplasm of B-cells of pancreas past staining by TSQ solution as by using of vital histochemical reaction past intravenous injection of TSQ to animals. We observed decreasing intensity of fluorescence past partial and almost complete removing of Zn+2-ions off B-cells by 3 days prolonged per oral treatment by Glibenclamide, 15–20 mg/kg daily [Fig. 1.2, 1.3].Results of fluorescent microscopy of sections demonstrate partial or almost complete negative reaction for Zn+2-ions — as result of removing of Zn+2-ions from B-cells [Fig. 1.3]. Negative fluorescent reaction for Zn+2-ions in islets of animals with diabetes caused by selective destruction of B-cells by Dithizone (Fig. 1.5, 1.6; Tables 1, 2).
Figure 1
We observed intensive fluorescence of cytoplasm contained a large amount Zn+2-ions cells of Prostate and Salivary gland (Fig. 1.8, 1.9) past intravenous injection of 8PTSQ solution.
Animals with diabetes caused by injection of DZ (50,2 mg/kg). Negative reaction for Zn+2-ions with 8PTSQ as for insulin in B-cells in sections of pancreas tissue (fig. 3, 2.4; control 2.1, 2.2; Table 1, 2) that demonstrate absence in cytoplasm of B-cells as of Zn+2-ions as of insulin in result of necrosis and destruction of cells: Rabbits: K(IF1/IF2)=1,03±0,05; control: intact B-cells: K=2,06±0,07 (p<0,001); Mice: 1,89 ±0,06 and control (intact)=1,06±0,04.Insulin content in B-cells: K(IG1/IG2)=1,12 ±0,03; intact B-cells IG1/IG2=1,92±0,04 (Table 1, 2).
Figure 2. Zinc-ions and insulin content in B-cells of intact and experimental rats
Zn+2-ions content in B-cells (parameter K: IF1/IF2)
T a b l e 1
Insulin and Zinc content in pancreatic B-cells (parameter K)
T a b l e 2
Results showed that in 3 cases method demonstrated a full coincidence of Zn+2-ions content with content of insulin in B-cells: 1) in intact animals; 2) in animals with experimental diabetes; 3) in animals after removing of Zn+2-insulin complex from B-cells by drugs.
This method demands following conditions. For fixation of tissue of pancreas to use the 70º alcohol sated with hydrogen sulfide (H2S) or to use sections of frozen-pancreas tissue. Filters for UV-microscopy: UV-filter between UV-lamp and microscope and yellow filter for ocular of microscope. 8PTSQ is high specific fluorescent reagent for revealing of minimal concentrations of Zn+2-ions in solutions as 10–7–10–8.
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