Histochemical method for fluorescent staining of Zn+2-ions in glands

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A.A.Kikimbaeva

^{1, 4}

, Z.T.Kystaubaeva

^{1, 2}

, G.M.Tykezhanova

^{1, 2}

, L.K.Bystrevska

³

, A.G.Abdraimova-Meyramova

^{1, 3}

, Ye.M.Laryushina

³

, S.B.Zhautikova

³

, V.N.Beskov

³

,

G.O.Zhuzbaeva

²

, A.R.Alina

³

, S.S.Turzhanova

²

, G.K.Turlibekova

²

1Diabetes Research Group, Karaganda;

2Ye.A.Buketov Karaganda State University;

3Karaganda State Medical University;

4Astana Medical University (E-mail: meyramow@mail.ru)

Histochemical method for fluorescent staining of Zn

⁺²

-ions in glands

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 sensi- tive and high specific for revealing of Zn⁺²-ions and there are only one step of staining procedures. Mean- while it is possible to use only fresh frozen sections of tissues for investigation within short time limited by 15–20 min.

Key words: B-cells, Prostate, Salivary glands, 8-para(toluenesulphonylamino)quinolone, Zn⁺²-ions.

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 zinc- insulin 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 ad- dition the zinc ions enhance proinsulin solubility and render insulin insoluble. Pancreas of animals as of Hu- man 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 nec- essary 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

1. 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 nanometers. 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);

2. 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 +70⁰Celsius and injected to Rabbits 36,5–38,8 mg/kg.

3. Staining of insulin and Zn⁺²-ions content in B-cells of animals with experimental diabetes caused by injection of Dithizone.

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1. Intact animals. Intensive fluo cytoplasm of B-cells was revealed i using of vital histochemical reaction intensity of fluorescence past partia longed per oral treatment by Gliben copy of sections demonstrate partia moving of Zn⁺²-ions from B-cells [F with diabetes caused by selective de

1.1

1.4

1.7

1.1 Intact Rabbit. Fluorescence of Zn⁺ 1.2 Injection of DDCA 250 mg/kg. Sta 1.3 Injection of DDCA 1000 mg/kg. S negative fluorescent reaction for Z 1.4 Injection of 8PTSQ 38,8 mg/kg; v

×140;

1.5 Rabbit, Destruction of B-cells cau pancreas; ×200;

1.6 Rabbit, Destruction of B-cells cau by 8PTSQ: absence of Zn⁺²-ions in 1.7 Intact Mice. Fluorescence of Zn⁺²- 1.8 Frozen section of Prostate tissue o 1.9 Frozen section of Salivary gland; S

Results

orescence of complex Zn⁺²-ions-TSQ as positive in cytoplasm of B-cells of pancreas past staining n past intravenous injection of TSQ to animals. W al and almost complete removing of Zn⁺²-ions of

clamide, 15–20 mg/kg daily [Fig. 1.2, 1.3].Resul al or almost complete negative reaction for Zn⁺² Fig. 1.3]. Negative fluorescent reaction for Zn⁺²- estruction of B-cells by Dithizone (Fig. 1.5, 1.6; T

1.2 1.3

1.5 1.6

1.8 1.9

+2-ions in B-cells. Frozen section of Pancreas. Staining aining by 8PTSQ; partial binding of Zn⁺²-ions in B-ce Staining by 8PTSQ; almost complete binding of Zn⁺²- Zn⁺²-ions in B-cells; ×140;

vital staining of Zn⁺²-ions in B-cells past injection. F used by injection of Dithizone, 48,6 mg/kg; dark mic used by injection of Dithizone, 48,6 mg/kg; frozen se

n B-cells;

-ions in B-cells. Frozen section of Pancreas. Staining b f Rabbit. Staining by 8PTSQ. Fluorescense of Zn⁺²-ion Staining by 8PTSQ. Fluorescence of Zn⁺²-ions; ×140

Figure 1

reaction for Zn⁺²-ions in g by TSQ solution as by We observed decreasing ff B-cells by 3 days pro- lts of fluorescent micros-

2-ions — as result of re- -ions in islets of animals Tables 1, 2).

g by 8PTSQ; ×140;

lls by DDCA; ×140;

-ions in B-cells by DDCA;

Frozen section of pancreas;

croscopy; frozen section of ection of pancreas, staining by 8PTSQ; ×140;

ns; ×140;

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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.

2. 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. 2.3, 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).

2.1 2.2

2.3 2.4 2.1 Pancreatic islet of intact rabbit. 8PTSQ fluorescent reaction for zinc. Intensive fluorescence (a large amount of

zinc in B-cells); UV-light microscopy; ×140;

2.2 Pancreatic islet of intact rat. Immunohistochemical method. Normal content of deposited insulin in B-cells (blue- violet color); ×280;

2.3 Pancreatic islet of rat with dianetes. 8PTSQ fluorescent reaction for zinc. Negative reaction for zinc (absence of fluorescence) determined by destruction of B-cells and by absence of zinc-ions in cytoplasm; UV-light microscopy; ×140;

2.4 Panc reatic islet of rat with diabetes. Immunohistochemical staining method. Decreasing of insulin content in B-cells and of size and number of islets in sections; ×280

Figure 2. Zinc-ions and insulin content in B-cells of intact and experimental rats

T a b l e 1 Zn⁺²-ions content in B-cells (parameter K: IF1/IF2)

№ Animals Intact animals (IF1/IF2)

Diabetes caused by Dithizone (IF1/IF2)

1 Rabbits 2,06±0,07^● 1,03±0,05^●

2 Rats 1,94±0,05 –

3 Mice 1,89±0,06* 1,06±0,04*

Note. ^● * — p<0,005.

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T a b l e 2 Insulin and Zinc content in pancreatic B-cells (parameter K)

№ Conditions of experience Insulin (IG) and Z+2 content (IF) in B-cells (parameter K) insulin (IG) zinc (IF)

1 5 min. past injection of DZ 1,88±0,05 1,03±0,05

2 Diabetes caused by DZ (48,8–51,6 mg/kg) 1,12±0,03^● 1,08±0,03

3 DDCA (987 mg/kg) 1,85±0,04 1,02±0,04^*

5 Rabbit (intact) 1,92±0,04^● 1,98±0,06^*

Note. ^● * — p<0,001.

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 sat- ed 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.

References

1 Eisebrandt J., Scienz M., Wegel F. Uber den Zinkgehalt menschicher Pancreasdrussen und uber das Bindungs Vormogen des Insulins fur Zink // Medizinund Chemie. — 1942. — No. 8. — P. 259–296.

2 Schmidt R., Rautschke R. Der Zink und Kurfergehalt verschiedener Organe der weissen Ratte: ein Beitrag zur Dithizon-Zink- Reaction in der Histochemie // Acta Histochem. — 1964. — Vol. 19. — P. 1–13.

3 Galabova R., Petkov P., Kolev J. Rontgen-Fluoreszenz Untersuchung von Zink, Kupfer und Kobalt in Pancreas einiger San- ger // Acta Histochem. — 1971. — No. 2. — P. 335–342.

4 Andersson T., Betgreen P., Flatt P. Subcellular distribution of zinc in islets B-cells fractions // Hormones and Metabolism Res. — 1980. — Vol. 12, No. 1. — P. 275–276.

5 Emdin S.O., Dodson G.G., Cutfield J.M., Cutfield S.M. Role of zinc in insulin biosynthesis. Some possible zinc-insulin inter- actions in the pancreatic B-cell // Diabetologia. — 1980. — Vol. 19, No. 3. — P. 174–82.

6 Okamoto K. Experimental production of diabetes // Diabetes mellitus: Theory and Practice. — New-York: McGraw-Hill Book company, 1970. — P. 230–255.

7 Красавин И.А., Бавельский З.Е., Лазарис Я.А., Дзиомко В.М. Гистохимические реакции на цинк в островках Лангер- ганса и диабетогенная активность используемых веществ // Проблемы эндокринологии. — М., 1969. — № 3. — С. 102–105.

8 Божевольнов Е.А., Серебрякова Г.В. 8-пара(толуолсульфониламино)хинолин — люминесцентный реагент на цинк и кадмий // Химические реактивы и препараты. — М., 1961. — С. 36–42.

А.А.Қиқымбаева, З.Т.Қыстаубаева, Г.М.Тыкежанова, Л.К.Быстревская, А.Ғ.Əбдірайымова-Мейрамова, Е.М.Ларюшина, С.В.Жəутікова, В.Н.Бесков,

Г.Ө.Жүзбаева, А.Р.Алина, С.С.Тыржанова, Г.Қ.Тұрлыбекова

Zn

⁺²

иондарын бездерде флюоресцентті анықтаудың гистохимиялық əдісі

Авторлармен 8-пара(толуолсульфониламино)хинолин — 8-оксихинолиннің туындысын қолдану флюоресценттік микроскопияның көмегімен қуықалды безінің ұлпа жасушасында, панкреатикалық В-жасушасында жəне сілекей бездерінде Zn⁺² иондарын айқындауға мүмкіндік беретіндігі көрсетілген. Əдістің өзгешілігі — оның жоғары сезімталдығы, жасушаларда Zn⁺² иондарын айқындау ерекшелігі жəне боялу тəртібінің бірсатылығы. Салыстырмалы түрдегі кемшілігі — тұрақты гистологиялық препараттарды алу мүмкіндігінің болмауы. Препараттар аз уақыт сақталады — 15–20 мин.

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А.А.Кикимбаева, З.Т.Кыстаубаева, Г.М.Тыкежанова, Л.К.Быстревская, А.Г.Абдраимова-Мейрамова, Е.М.Ларюшина, С.В.Жаутикова, В.Н.Бесков,

Г.О.Жузбаева, А.Р.Алина, С.С.Тыржанова, Г.К.Турлыбекова

Гистохимический метод флюоресцентного выявления ионов Zn

⁺²

в железах

Авторами показано, что использование 8-пара(толуолсульфониламино)хинолина — производного 8-оксихинолина позволяет с помощью флюоресцентной микроскопии выявлять ионы Zn⁺²в клетках ткани предстательной железы, в панкреатических В-клетках и в слюнных железах, где он содержится в значительных количествах. Отличительная особенность метода — его высокая чувствительность, абсолютная специфичность в отношении выявления ионов Zn⁺² в клетках и одноэтапность процедуры окраски. Относительный недостаток состоит в отсутствии возможности получения постоянных гисто- логических препаратов. Препараты сохраняются относительно недолго — в течение 15–20 мин.

References

1 Eisebrandt J., Scienz M., Wegel F. Medizinund Chemie, 1942, 8, p. 259–296.

2 Schmidt R., Rautschke R. Acta Histochem., 1964, 19, p. 1–13.

3 Galabova R., Petkov P., Kolev J. Acta Histochem., 1971, 2, p. 335–342.

4 Andersson T., Betgreen P., Flatt P. Hormones and Metabolism Res., 1980, 12, 1, p. 275–276.

5 Emdin S.O., Dodson G.G., Cutfield J.M., Cutfield S.M. Diabetologia, 1980, 19, 3, p. 174–82.

6 Okamoto K. Diabetes mellitus: Theory and Practice, New York: McGraw-Hill Book company, 1970. — p. 230–255.

7 Krasavin I.A., Bavelsky S.E., Lazaris Y.A., Dziomko V.M. Problems of Endocrinology, Moscow, 1969, 3, p. 102–105.

8 Bogevolnov E.A., Serebrakova G.V. Chemicals and drugs, Moscow, 1961, P. 36–42.

Acknowlegement

Gratitude to Prof. G.G.Meyramov for financing of investigation.