Биологические науки / 8. Физиология человека и животных

Grigorova N.V., Psol M.A.

Zaporizhzhya National University

THE STATE OF BLOOD GRANULOCYTES AND PANCREATIC ISLETS OF GOLDEN HAMSTERS UNDER VARIOUS FUNCTIONAL STATE OF INSULAR APPARATUS

Zinc performs important biological functions in a living organism. It is necessary for activation of more than 200 metalloenzymes, maintains integral structure and function of biological membranes [1, 6-8]. In pancreatic beta cells two zinc ions are able to bind six insulin molecules resulting in hexamer forming [2, 4, 5]. Hexamer is considered a deposit form of this hormone [3]. Blood granulocytes and pancreatic islands contain cytochemically determinate zinc which gives us an opportunity to suppose a presence of functional connection between them[3]. For this purpose golden hamsters were exposed to the influence of the factors which altered functional state of insular apparatus: starvation inhibited incretory function of insular apparatus, glucose load specifically stimulated secretion of insulin, injection of diabetogenic agent alloxan removed activity of insular apparatus. The content of zinc was determined simultaneously with the level of insulin in pancreatic beta cells and secretory material in blood granulocytes for the confirmation of the role of this microelement as a depositing agent.

40 golden hamsters were used in experiments, including 10 control (intact) animals and those subjected to the experimental influences. The animals starved during 24 hours. 40% solution of glucose was injected intraperitoneally in the dose of 10 g/kg. 24% solution of alloxan was injected subcutaneously in the dose of 200-400 mg/kg. Blood probes were taken from the tail after the ending of starvation period, in 2 hours after glucose infusion and in 5 days after alloxan injection. Then animals were decapitated and pancreas pieces were taken for preparation of the organ sections.

Before cytochemical reactions blood smears were fixed during 5 minutes in ascending formalin vapour and were stained with 0,2% water-ammoniac solution of dithizone. Time of fixation was 3 hours. Red granules were revealed in cytoplasm of blood granulocytes. Their amount is an indicator of content of zinc in the cells.

For the cytochemical investigation of secretory material in granular leukocytes blood smears were sequentially stained in 1% water solutions of methyl green and eosine during 30 minutes. On the slides violet granules were revealed in neutrophils, red ones in eosinophils and blue ones in basophiles.

Pieces of pancreas were fixed in cold acetone during 12 hours (for cytochemical determination of zinc in pancreatic islets) and in Bowin’s fluid during 24 hours under the room temperature (for cytochemical determination of insulin). Fixed in acetone pieces were run through xylols, mix of xylols and paraffin, liquid paraffins and were embedded in paraffin. Pieces fixed in Bowin’s fluid were embedded in paraffin.

Paraffin sections were prepared 5-10 μm thick. Sections prepared from the acetone fixed pieces were deparaffinized by carrying out through xylols, acetones, distilled water. Then they were treated with 0,01% acetone solution of 8-(p-toluensulfonylamino)–quinoline (8-TSQ) and examined with luminescent microscope. Yellow-green luminescent granules were revealed in cytoplasm of pancreatic beta cells on the slides. Intensity of luminescent reaction served as an indicator of zinc content in these cells.

Deparaffinized sections of pancreas fixed in Bowin’s fluid sequentially were carried out through oxidizer, reducer, and stained with aldehyde fuchsin. On the slides blue-violet granules were revealed in pancreatic B cells. Amount of granulation is an indicator of insulin content in the cells.

Intensity of cytochemical reactions was evaluated with semiquantitative method. One point was given for a weak positive reaction, two points for a moderate one, and three points for a highly intensive reaction. According to calculation on 100 cells we estimated arithmetic mean value (), error (m), probability (p).

In blood granulocytes of control (intact) golden hamsters intensity of reaction for dithizone was 1,0 + 0,08 conditional units (c.u.), for methyl green and eosine was 0,9 + 0,08 c.u.

Intensity of dithizone reaction in granular leucocytes raised on 40% (1,4 + 0,09 c.u.; p < 0,01) after starvation, decreased on 30% (0,7 + 0,07 c.u.; p < 0,02) after glucose injection, dropped on 40% (0,6 + 0,07 c.u,; p < 0,01) after alloxan injection.

Intensity of methyl green and eosine reaction in granulocytes increased on 33% (1,2 + 0,08 c.u.; p < 0,01) as a result of acute starvation, dropped on 22% (0,7 + 0,03 c.u.; p < 0,05) after glucose injection, declined on 44% (0,5 + 0,04 c.u.; p < 0,001) in animals treated with alloxan.

In the pancreatic B cells of control (intact) golden hamsters intensity of 8-TSQ reaction was 1,6 + 0,09 c.u., of aldehyde fuchsin reaction – 1,4 + 0,07 c.u.

Starvation of animals caused increasing of zinc content in the pancreatic B cells on 31% (2,1 + 0,13 c.u.; p < 0,01) and increasing of insulin concentration on 21% (1,7 + 0,09 c.u.; p < 0,02). Glucose load caused declining of zinc on 19% (1,3 + 0,05 c.u.; p < 0,02) and insulin on 14% (1,2 + 0,04 c.u.; p < 0,05) in pancreatic B cells of golden hamsters. After injection of alloxan concentration of zinc in islets decreased on 81% (0,3 + 0,04 c.u.; p < 0,001), content of insulin dropped on 86% (0,2 + 0,01 c.u.; p < 0,001).

Thus, weakening of incretory function of pancreas under starvation is accompanied by accumulation of zinc and secretory material in blood granulocytes, zinc and insulin in pancreatic B cells of hamsters. Activation of insular apparatus function as a result of glucose load caused declining of the researched compounds content in cells. Deficits of zinc, secretory material and insulin in blood granulocytes and pancreas were observed after the injection of diabetogenic agent alloxan, which selectively damages insulin producing cells. Received data confirm existence of functional connection between blood granulocytes and B cells of pancreas.

References:

1. Bettger W.Y., O’Dell L.L. A critical physiological role of zinc in the structure and function of biomembranes // Life Sci. -1981. –Vol.28. –P. 1405 - 1438.

2. Chausmer A.B. Zinc, insulin and diabetes // J. Am. Coll. Nutr. -1998. –Vol.17, №2. –P. 109 - 115.

3. Grigorova N.V., Eshchenko J.V., Bovt V.D., Eshchenko V.A. Cytochemical zinc investigations // Materials of III International scientific practical conference “Actual questions of modern science: theory and practice”. – 2006. – Vol.2. – Dnepropetrovsk: Science and education, 2006. – P. 27 - 29.

4. Rosenfeld L. Insulin: discovery and controversy // Clin. Chem. – 2002. –Vol.48, № 12. –P. 2270 - 2288.

5. Seale A.P., de Jesus L.A., Kim S.J., Choi J.H., Lim H.B., Hwang C.S., Kim J.S. Development of an automated protein-tyrosine phosphotase 1B inhibition assay and the screening of putative insulin-enhancing vanadium (IV) and zinc (II) complexes // Biotechnol. Lett. - 2005. –Vol.27, №4. –P. 221 - 225.

6. Tudor R., Zaleski P.D., Ratnaike R.N. Zinc in health and chronic disease // J. Nutr. Health Aging. -2005. –Vol.9, №1. –P. 45 – 51.

7. Varvarra G., Traini T., Esposito P., Caputi S., Perinetti G. Copper-zinc superoxide dismultase activity in healthy and inflamed human dental pulp // Int. Endod. J. -2005. –Vol.38, №3. –P. 195 -199.

8. Vitek L., Muchova L., Zelenka J., Zadinova M., Malina J. The effect of zinc salts on serum bilirubin levels in hyperbilirubinemic rats // J. Pediatr. Gastroenterol. Nutr. -2005. –Vol.40, №2. –P. 135-140.