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Etape

 

I. INTERMEDIARY PERIOD (month 1 To Month 12) ( 12 months )

Obiective


Activitati

1. At the Coordinator Institution The Pharmacology Department, Faculty of Medicine, University of Medicine and Pharmacy Craiova Romania,

It will be used 10 (ten) strips of rat uteri for each type of determination.
Experiments will be performed on non-pregnant and pregnant myometrium preleaved from female rats. For the first group, animals selected will be in diestrum period, established through vaginal smear. For the second group it will be used 3 weeks pregnant female rats.
For all experiments it will be asked the USA & Romanian University Laboratory Animal Care Committee Agreement.
The determinations will be done in Krebs-Henseleit solution, oxygenated with O2 95% and CO2 5%, thermostated at 37oC, into an organ bath (AD Instruments).

Experiments performed will study, separately for each of the two types of myometrium non-pregnant and pregnant:

a. effect of G36 a GPER specific agonist in different concentrations (10-4M, 10-5M, 10-6M, 10-7M, 10-8M) on spontaneously contractile activity of rat myometrium and select the optimal concentration for future determination, if it will be obtained any effect;

b. effect of estradiol in different concentrations (10-4M, 10-5M, 10-6M, 10-7M, 10-8M) on spontaneously contractile activity of rat myometrium and select the optimal concentration for future determination, if it will obtained any effect;

c. effect of several compounds which interfere with calcium channels on GPER stimulation with G36, on uterine strips, if it will be obtained any effect with G36 alone:
- nifedipine membranal calcium channels type L inhibitor,
- ryanodine ryanodine-sensitive calcium channels inhibitor,
- diethyl phenyl amino borate IP3-sensitive calcium channels inhibitor,
- bafilomicyn lisosomal calcium stores destroyer.

2. At the Scientific Consultant and Technical Support Laboratory The Pharmacology Department, School of Medicine, Temple University, Philadelphia USA,

a. Finding the best protocols for obtaining cell cultures from rat myometrium smooth muscle, from both diestrum and 3 weeks pregnant female rats.

b. Proving the presence of GPER in uterine non-pregnant and pregnant smooth muscle culture cells, by PCR techniques.

c. Establishing the technique of intracellular administration of substances in uterine smooth muscle cells.

II. FINAL PERIOD (month 13 To Month 24) ( 12 months )

Obiective


Activitati

1. At the Coordinator Institution The Pharmacology Department, Faculty of Medicine, University of Medicine and Pharmacy Craiova Romania,

In case of significant results on rat uterine strips, it will be remade all experiments on human myometrium fragments, preleaved, only after inform consent.
It will be used non-pregnant myometrium preleaved during myomectomy or hysterectomy for uterine fibromatosis and pregnant myometrium fragments obtained during cesarean operation or needed hysterectomy during delivery.


2. At the Scientific Consultant and Technical Support Laboratory The Pharmacology Department, School of Medicine, Temple University, Philadelphia USA,

In case of significant results obtained by the Coordinator Institution, represented by the Pharmacology Department, Faculty of Medicine, University of Medicine and Pharmacy Craiova Romania, during intermediary period, it will be used micro organ bath and adapted fluorescent techniques for measurement of intracellular calcium concentrations, in uterine smooth muscle culture cells.

a. It will be measured the G36 effect, in different concentrations (10-4M, 10-5M, 10-6M, 10-7M, 10-8M), on intracellular calcium mobilization;

b. The effect of estradiol on intracellular calcium mobilization, in the same concentrations, will be used as martor.

c. It will be studied the effect of intracellular administration of the following compounds on G36-induced intracellular calcium mobilization, if any significant effect will be obtained with G36 alone:
- ryanodine ryanodine-sensitive calcium channels inhibitor,
- xestospongine C IP3-sensitive calcium channels inhibitor,
- bafilomicyn lisosomal calcium stores destroyer,

d. It will be studied the effect of intracellular administration of the following compounds, on estradiol-induced intracellular calcium mobilization, if any significant effect with estradiol alone will be obtained:
- ryanodine ryanodine-sensitive calcium channels inhibitor,
- xestospongine C IP3-sensitive calcium channels inhibitor,
- bafilomicyn lisosomal calcium stores destroyer,

e. Effect of nifedipine membranal calcium channels type L inhibitor, on G36 -induced intracellular calcium mobilization, if any significant effect will be obtained by using G36 alone.

f. Effect of nifedipine membranal calcium channels type L inhibitor, on estradiol-induced intracellular calcium mobilization, if any significant effect will be obtained by using estradiol alone.


All Consortium Institutions:

Integrated analysis of the results, with special attention on differences (if there will be some) between:
- isolated myometrium cells and uterine fragments;
- pregnant and non-pregnant myometrium.