Phenytoin teratogenicity: hypoxia marker and effects on embryonic heart rhythm suggest an hERG-related mechanism.

BACKGROUND: The antiepileptic drug phenytoin (PHT) is a human and animal teratogen. The teratogenicity has been linked to PHT-induced embryonic cardiac arrhythmia and hypoxic damage during a period when regulation of embryonic heart rhythm is highly dependent on a specific K(+) ion current (I(Kr)). PHT has been shown to inhibit I(Kr). The aims of this study were to investigate whether teratogenic doses cause embryonic hypoxia during and after the I(Kr) susceptible period and to further characterize PHT effects on embryonic heart rhythm. METHODS: Pregnant C57BL mice were administered the hypoxia marker pimonidazole followed by PHT or saline (controls) on GD 10 or GD 15. The embryos were fixed and sectioned, and the immunostained sections were analyzed with a computer assisted image analysis. Effects of PHT (0-250 microM) on heart rhythm in GD 10 embryos cultured in vitro were videotaped and then analyzed by using a digitalization technique. RESULTS: PHT dose-dependently increased the hypoxia staining (6- and 11-fold after maternal dosing of 100 and 150 mg/kg, respectively) during the period I(Kr) is expressed and functional (GD 10). In contrast, there were no differences between the PHT doses in hypoxia staining, and much less pronounced hypoxia after this period (GD 15). With increasing PHT concentrations, increased length of the interval (bradycardia) and large variations in length between individual heartbeats (arrhythmia) were recorded. CONCLUSIONS: PHT induced bradycardia/arrhythmia and severe embryonic hypoxia during the I(Kr) susceptible period, supporting the idea of an I(Kr)-arrhythmia-hypoxia-related teratogenic mechanism.

Teratogenicity by the hERG potassium channel blocking drug almokalant: use of hypoxia marker gives evidence for a hypoxia-related mechanism mediated via embryonic arrhythmia.

The rapid component of the delayed rectifying potassium ion current (IKr), plays an important role in cardiac repolarization. In rats, potent IKr channel blocking drugs cause similar stage-specific malformations (such as orofacial clefts and digital reductions) on gestational days (GDs) 10-14 as after periods of embryonic oxygen deprivation (hypoxia). The idea of a hypoxia-related teratogenic mechanism is supported by studies using rat embryos cultured in vitro. These studies show that the embryonic heart reacts with concentration-dependent bradycardia, arrhythmia, and cardiac arrest when exposed to IKr blockers on GDs 10-14. The main purpose of this study was to investigate whether previously shown teratogenic doses on GD 11 and 13 of the selective IKr blocker almokalant (ALM) induce hypoxia in rat embryos in vivo by using the hypoxia marker pimonidazole hydrochloride (PIM). Rats were orally dosed with almokalant or tap water on GD 11 (150 micromol/kg), 13 (50 micromol/kg), or 16 (800 micromol/kg), followed by PIM intravenously 30 min later. Two hours after the PIM dose, the embryonic heart activity was videotaped and analysed, and the embryos were fixed, sectioned, and immunostained. Computer-assisted image analysis showed a two- and threefold increase in hypoxia staining in embryos exposed to teratogenic doses of ALM on GDs 11 and 13. Embryonic arrhythmia was observed in almokalant groups on these GDs, but not in controls. In contrast, dosing on GD 16, with a much higher dose (800 micromol/kg), caused neither hypoxia nor any effects on heart rhythm. The results support the IKr-related arrhythmia-hypoxia hypothesis, by showing that the potent IKr-blocking drug, almokalant, (1) causes severe embryonic hypoxia and arrhythmia at stages (GDs 11 and 13) when developmental toxicity could be induced and IKr is functional and (2) does not cause hypoxia or affect heart rhythm at a developmental stage when IKr is suppressed (GD 16) and potent IKr blockers do not induce developmental toxicity.

Purinergic receptor signaling at the basolateral membrane of macula densa cells.

Purinergic receptors are important in the regulation of renal hemodynamics; therefore, this study sought to determine if such receptors influence macula densa cell function. Isolated glomeruli containing macula densa cells, with and without the cortical thick ascending limb, were loaded with the Ca(2+) sensitive indicators, Fura Red (confocal microscopy) or fura 2 (conventional video image analysis). Studies were performed on an inverted microscope in a chamber with a flow-through perfusion system. Changes in cytosolic calcium concentration ([Ca(2+)](i)) from exposed macula densa plaques were assessed upon addition of adenosine, ATP, UTP, ADP, or 2-methylthio-ATP (2- MeS-ATP) for 2 min added to the bathing solution. There was no change in [Ca(2+)](i) with addition of adenosine (10(-7) to 10(-3) M). UTP and ATP (10(-4) M) caused [Ca(2+)](i) to increase by 268 +/- 40 nM (n = 21) and 295 +/- 53 nM (n = 21), respectively, whereas in response to 2MesATP and ADP, [Ca(2+)](i) increased by only 67 +/- 13 nM (n = 8) and 93 +/- 36 nM (n = 14), respectively. Dose response curve for ATP (10(-7) to 10(-3) M) added in bath showed an EC(50) of 15 microM. No effect on macula densa [Ca(2+)](i) was seen when ATP was added from the lumen. ATP caused similar increases in macula densa [Ca(2+)](i) in the presence or absence of bath Ca(2+) and addition of 5 mM ethyleneglycotetraacetic acid (EGTA). Suramin (an antagonist of P2X and P2Y receptors) completely inhibited ATP-induced [Ca(2+)](i) dynamics. Also, ATP-Ca(2+) responsiveness was prevented by the phospholipase C inhibitor, U-73122, but not by its inactive analog, U-73343. These results suggest that macula densa cells possess P2Y(2) purinergic receptors on basolateral but not apical membranes and that activation of these receptors results in the mobilization of Ca(2+).