*3.1.3 PCR studies*

*Antioxidants*

**3. Results**

*3.1.1 Sodium flux studies*

*3.1.2 Immunoblotting*

middle cerebral artery occlusion studies.

Euthanasia of the American Veterinary Medical Association.

mil inhibitable cationic channel (Kd = 1.7 μl/mg/min).

*sensitive (100 μM), indicative of a sodium-hydrogen ion exchanger.*

**3.1 Evidence for the presence of NHE1, NCX1, and Na/K ATPase**

Health and Science University (OHSU), Portland, OR. OHSU is in full compliance with Federal and State statutes regarding the use and care of vertebrate animals in research. The animal care facility is AAALAC accredited, and all rat maintenance, treatment, recovery, and euthanasia procedures were approved for the reported

To achieve middle cerebral artery occlusion, female Long Evans rats (250–300 grams) were anesthetized with isoflurane inhalant (5% induction, 2% maintenance). Analgesia was provided by administering butorphanol (0.05–2.0 mg/kg SQ ) as needed, and seizures were controlled with diazepam (5 mg/kg) if necessary. Animals were monitored daily by staff veterinarians. Upon completion of the experiments, the rats were euthanized after inhalant anesthetic induction with an intracardiac overdose of sodium pentobarbital, or if symptoms necessitated earlier sacrifice. This method of euthanasia is consistent with requirements of the Panel of

22Na uptake was measured in membrane vesicles from brain capillary endothelial cells, and revealed both saturable and unsaturable components (**Figure 1**). In the presence of specific inhibitors, three transport pathways were found, including: (a) a low affinity (Km = 52 mM), high capacity, dimethyl amiloride (DMA, 100 μM) sensitive sodium carrier, indicative of Na/H antiport; (b) a high affinity (Km = 4.6 mM), low capacity, DMA resistant carrier; and (c) a non-specific phena-

Immunoblotting using specific antibodies (Chemicon) to NHE1 (Na/H antiporter, isoform 1) and NCX1 (Na/Ca exchanger, isoform 1) revealed that both proteins are present in isolated luminal and abluminal membranes derived from

*Both saturable and unsaturable 22Na uptake was measured in plasma membrane vesicles derived from bovine brain capillary endothelial cells. A saturable component (Km = 52 mM) was dimethyl amiloride (DMA)* 

**202**

**Figure 1.**

Messenger RNA was prepared from isolated bovine brain capillaries, and RT-PCR was performed with specific primers identifying NHE1, Na/K ATPase α2, and Na/K ATPase α3. The expected PCR products were found for NHE1 (**Figure 2**) and Na/K ATPase α3, and sequencing demonstrated a high level of homology to the human transporters (>90%). Na/K ATPase activity was previously measured by us in luminal and abluminal membrane vesicles derived from endothelial cells forming the blood-brain barrier [25]. Activity was found to be present predominantly at the abluminal membrane domain, and was characterized by a ouabain binding constant (Kd = 25 ± 3 nM) typical of either the α2 or α3 isoform [49]. The PCR studies reported in the current study confirmed that it is the α3 isoform.
