Efekat otvarača kalijumovih kanala pinacidila na tonus izolovane humane umbilikalne vene porodilja sa gestacijskim dijabetesom i hipertenzijom

Abstract

Humana umbilikalna vena je specifičan krvni sud koji se nalazi pod uticajem brojnih metaboličkih, vaskularnih i hemodinamskih promena tokom čitavog fetalnog života. Pinacidil je otvarač kalijumovih kanala (K kanala) čije je mesto vezivanja lokalizovano na SURx subjedinici KATP kanala. Osim toga, postoje i mnogi dokazi da pinacidil, primenjen u visokim dozama (≥ 100 μM), pored KATP kanala deluje i na druge podtipove K kanala, uključujući Kv i BKCa kanale, kao i da ima dejstva nezavisna od K kanala. U brojnim istraživanjima pokazano je da dijabetes melitus i hipertenzija uzrokuju promene u ekspresiji i funkciji K kanala u vaskularnim glatkim mišićnim ćelijama različitih krvnih sudova i kod različitih vrsta. Sa druge strane, opisano je da gestacijski dijabetes melitus (GDM) i trudnoćom izazvana hipertenzija (PIH) uzrokuju različite morfološke i ultrastrukturalne promene na humanim umbilikalnim venama. Ciljevi ovog istraživanja su bili: 1) da se ispita da li se ekspresija subjedinica nekoliko podtipova K kanala (KATP, BKCa i Kv) na humanim umbilikalnim venama zdravih porodilja razlikuje u odnosu na porodilje sa GDM i PIH; 2) da se ispita postoje li razlike u efikasnosti i potentnosti pinacidila u izazivanju vazodilatacije prekontrahovanih venskih segmenata zdravih porodilja i porodilja sa GDM i PIH; 3) da se definišu podtipovi K kanala uključeni u vazodilatatorne efekte pinacidila u svim grupama; 4) da se utvrdi da li pinacidil ima dejstva nezavisna od aktivacije K kanala. Ukupno 72 porodilje su bile uključene u studiju. Njih 40 je bilo zdravo, 16 je imalo GDM i 16 PIH. Nakon preparacije i izolacije humanih umbilikalnih vena iz pupčanika, venski segmenti su ili bili čuvani u 10% formaldehidu za imunohistohemijsko bojenje, zamrzavani na -70°C za Western blot analizu ili montirani u kupatilo za izolovane organe u cilju sprovođenja farmakoloških eksperimenata. Humana umbilikalna vena je kontrahovana serotoninom. Rastuće koncentracije pinacidila (0.1 – 1000 μM) dodavane su u kupatilo na kumulativan način. Za proveru umešanosti različitih tipova K kanala u vazorelaksaciji izazvanoj pinacidilom sledeći antagonisti dodavani su u kupatilo 20 minuta pre serotonina: glibenkamid (GLB) – specifični blokator KATP kanala; iberiotoksin – selektivni antagonista BKCa kanala; 4-aminopiridin (4-AP) – neselektivni antagonista Kv kanala; margatoksin – specifični antagonista Kv1.2 i Kv1.3 kanala i friksotoksin – specifični antagonista Kv4.2 i Kv4.3 kanala. Za ispitivanje mehanizama dejstva pinacidila nezavisnih od K kanala druga kontrakcija prouzrokovana je rastvorom visoke koncentracije K+ (100 mM K+)...

metabolic, vascular and hemodynamic factors during entire fetal life. Pinacidil is potassium channel (K channel) opener whose binding site is well characterized and it is located on the SURx subunit of KATP channel. There are studies suggesting that pinacidil, especially in high doses (≥ 100 μM), in addition to KATP channels opens other subtypes of potassium channels, including BKCa and Kv channels, and that pinacidil also has K channel-independent mechanisms of action. Many studies have revealed alterations in the expression and function of K channels during diabetes mellitus and hypertension in vascular smooth muscle cells in other vascular beds and in different species. On the other hand, it has been described that gestational diabetes mellitus (GDM) and pregnancy-induced hypertension (PIH) cause various morphological and ultrastructural abnormalities in human umbilical veins. The aims of this research were: 1) to analyze is the expression of several K channels (KATP, BKCa and Kv) subunits in human umbilical veins from normal pregnancy different in comparison to GDM and PIH groups; 2) to test if there are differences in the potency and efficiency of pinacidil in inducing vasorelaxation of precontracted vein segments from normal pregnancy and GDM and PIH; 3) to define types of K channels involved in pinacidil-induced vasodilation on isolated human umbilical veins in all groups; 4) to determine if pinacidil possess K channel-independent mechanisms of actions. A total of 72 patients were engaged in the study. Out of them 40 were healthy, 16 had GDM and 16 PIH. After the isolation and preparation, human umbilical vein segments were either immersed in 10% formalin for immunohistochemical staining, frozen at -70°C for Western blot analysis or mounted in the organ bath for pharmacological experiments. Human umbilical veins were precontracted with serotonin. The increasing concentrations of pinacidil (0.1 – 1000 μM) were added to the bath cumulatively. To test the roles of several types of K channels in pinacidil-induced vasorelaxation, following antagonists were added to the bath 20 minutes before serotonin: glibenclamide (GLB) – a specific blocker of KATP channels; iberiotoxin – a selective blocker of BKCa channels; 4-aminopiridine (4-AP) – a nonselective blocker of Kv channels; margatoxin – a selective blocker of Kv1.2 i Kv1.3 channels and phrixotoxin – a selective blocker of Kv4.2 i Kv4.3 channels. In order to examine K-independent actions of pinacidil the second contractions were produced by K+-rich Krebs-Ringer solution (100 mM K+)...