Effects of epoetin alfa on the central nervous system.

Erythropoietin (EPO) is a glycoprotein that has been shown to mediate response to hypoxia, and is most notably recognized for its central role in erythropoiesis. In a series of experiments using rodent models, the ability of systemically administered recombinant human erythropoietin (r-HuEPO, epoetin alfa) to cross the blood-brain barrier and affect the outcome of neuronal injury or cognitive function was evaluated. It was shown that EPO and EPO receptors are expressed at capillaries of the brain-periphery interface, and that systemically administered epoetin alfa crossed the blood-brain barrier. Compared with control animals, epoetin alfa significantly reduced tissue damage in an ischemic stroke model when administered 24 hours before inducing stroke, with significant protection still evident when epoetin alfa was administered 6 hours poststroke. Epoetin alfa reduced injury by blunt trauma when administered 24 hours before trauma, with a significantly smaller volume of tissue necrosis noted when compared with controls. The observation that epoetin alfa may reduce nervous system inflammation was confirmed when an experimental autoimmune encephalomyelitis model in which rats were shown to have significantly delayed onset and reduced severity of experimental autoimmune encephalomyelitis symptoms after treatment with epoetin alfa. Epoetin alfa also was shown to ameliorate the latency and severity of seizures, and significantly increase survival versus controls when exposed to kainate. These findings suggest future potential therapeutic uses for epoetin alfa beyond its current use to increase erythropoiesis.

Erythropoietin crosses the blood-brain barrier to protect against experimental brain injury.

Erythropoietin (EPO), recognized for its central role in erythropoiesis, also mediates neuroprotection when the recombinant form (r-Hu-EPO) is directly injected into ischemic rodent brain. We observed abundant expression of the EPO receptor at brain capillaries, which could provide a route for circulating EPO to enter the brain. In confirmation of this hypothesis, systemic administration of r-Hu-EPO before or up to 6 h after focal brain ischemia reduced injury by approximately 50-75%. R-Hu-EPO also ameliorates the extent of concussive brain injury, the immune damage in experimental autoimmune encephalomyelitis, and the toxicity of kainate. Given r-Hu-EPO's excellent safety profile, clinical trials evaluating systemically administered r-Hu-EPO as a general neuroprotective treatment are warranted.

Parathyroid hormone-related protein protects against kainic acid excitotoxicity in rat cerebellar granule cells by regulating L-type channel calcium flux.

The parathyroid hormone-related peptide (PTHrP) and PTH/PTHrP receptor genes are widely expressed in the CNS and both are highly expressed in the cerebellar granule cell. We have shown previously that PTHrP gene expression in granule cells is depolarization-dependent in vitro and is regulated specifically by Ca2+ influx via L-type voltage-sensitive calcium channels (L-VSCCs). Kainic acid induces long-latency excitotoxicity in granule cells via L-VSCC-mediated Ca2+ influx. Here, we show that PTHrP is just as effective as the L-VSCC blocker, nitrendipine (NTR), in preventing kainate excitotoxicity. A competitive inhibitor of PTHrP binding abrogates its neuroprotective effect. Both NTR and PTHrP decrease 45Ca2+ influx to the same degree. These findings suggest that PTHrP functions in an autocrine/paracrine neuroprotective feedback loop that can combat L-VSCC-mediated excitotoxcity.

Chronic hypoglycemia and diabetes impair counterregulation induced by localized 2-deoxy-glucose perfusion of the ventromedial hypothalamus in rats.

Previous studies have demonstrated that the ventromedial hypothalamus (VMH) plays a critical role in sensing and responding to systemic hypoglycemia. To evaluate the mechanisms of defective counterregulation caused by iatrogenic hypoglycemia and diabetes per se, we delivered 2-deoxy-glucose (2-DG) via microdialysis into the VMH to produce localized cellular glucopenia in the absence of systemic hypoglycemia. Three groups of awake chronically catheterized rats were studied: 1) nondiabetic (with a mean daily glucose [MDG] of 6.9 mmol/l) BB control rats (n = 5); 2) chronically hypoglycemic nondiabetic (3-4 weeks, with an MDG of 2.7 mmol/l) BB rats (n = 5); and 3) moderately hyperglycemic insulin-treated diabetic (with an MDG of 12.4 mmol/l) BB rats (n = 8). In hypoglycemic rats, both glucagon and catecholamine responses to VMH glucopenia were markedly (77-93%) suppressed. In diabetic rats, VMH 2-DG perfusion was totally ineffective in stimulating glucagon release. The epinephrine response, but not the norepinephrine response, was also diminished by 38% in the diabetic group. We conclude that impaired counterregulation after chronic hypoglycemia may result from alterations of the VMH or its efferent pathways. In diabetes, the capacity of VMH glucopenia to activate the sympathoadrenal system is only modestly diminished; however, the communication between the VMH and the alpha-cell is totally interrupted.

Parathyroid hormone-related protein markedly potentiates depolarization-induced catecholamine release in PC12 cells via L-type voltage-sensitive Ca2+ channels.

PTH-related protein (PTHrP) is a normal product of many excitable cells of the nervous and endocrine systems. Functions of PTHrP in these tissues are, however, currently unknown. Prior study has suggested that a relationship exists between PTHrP and the L-type voltage-sensitive Ca2+ channel (L-VSCC). For example, in cerebellar granule neurons PTHrP gene transcription is regulated by Ca2+ influx specifically through this channel. Amino-terminal PTHrP products signal via the widely expressed PTH/PTHrP receptor, which is linked to both protein kinase A and C. These second messengers are known modulators of L-VSCC conductance. To determine whether PTHrP can modulate L-VSCC function, we studied catecholamine secretion in a PC 12 clone expressing the PTH/PTHrP receptor but not PTHrP. We found that PTHrP(1-36) (100 nM) to be an ineffective secretagogue for resting cells, but its presence markedly potentiates secretion to K+ depolarization. The PTHrP-augmented catecholamine secretion depends entirely upon L-VSCC Ca2+ influx and rapidly inactivates. Similar effects were produced by (Bu)2cAMP but not by carbachol. These observations support the hypothesis that PTHrP can regulate L-VSCC conductance. In the normal adrenal medulla that expresses both PTHrP and its receptor, PTHrP may act in an autocrine/paracrine fashion to modify catecholamine secretion.

Transcripts of the transposon mariner are present in epileptic brain.

Mobile genetic elements termed transposons have been increasingly implicated in human disease. The small transposon mariner is widespread within non-vertebrate genomes and causes mutation by replication, excision, and insertion of itself without an RNA intermediate. We find that human DNA contains about 60 copies of this gene. Mariner transcripts are abundant in RNA prepared from sclerotic epileptic hippocampi. In contrast, typically no mariner-specific RNA is detected in non-sclerotic hippocampi from other epileptic patients or from autopsies. A complete but non-functional copy was obtained using rapid amplification of cDNA ends (RACE). This human mariner transcript is approximately 45% homologous to a functional counterpart active in Drosophila, with a coding region of 1035 bases flanked by 32 base inverted terminal repeats. The differential expression of mariner transcripts within sclerotic hippocampi suggests the probable activity of an autonomous element which by mutating critical genes could establish an epileptogenic substrate in the hippocampus.

Neurosarcoidosis presenting as hypopituitarism and a cystic pituitary mass.

We report a young woman with clinical hypopituitarism and systemic sarcoidosis involving the lung, gastrointestinal tract, and peripheral lymph nodes. Laboratory evaluation confirmed that cortisol, thyroid indices, insulin-like growth factor 1, follicle-stimulating hormone, luteinizing hormone, and estradiol levels were low, with a normal prolactin. Magnetic resonance imaging revealed a large cystic pituitary lesion compressing the optic chiasm and exhibiting rim but not hypothalamic enhancement. The differential diagnosis included cystic macroadenoma, Rathke's cleft cyst, craniopharyngioma, and simple cyst. A transsphenoidal procedure provided decompression and diagnosis: pathology was consistent with sarcoidosis. Postoperatively, the patient's neurosarcoid disease markedly worsened, requiring hypothalamic irradiation. To our knowledge, this is the first report of intracranial sarcoidosis presenting solely as a cystic pituitary mass. An awareness of this possibility is important to prevent inappropriate neurosurgical intervention and subsequent potential exacerbation of neurosarcoidosis.

Dynorphin and the kappa 1 ligand [3H]U69,593 binding in the human epileptogenic hippocampus.

The distribution of dynorphin (DYN), one of its binding sites (kappa 1 receptor) and their relationship to neuronal loss and granule cell hyperexcitability was examined in hippocampi from patients with temporal lobe epilepsy (TLE). In hippocampi that were not the seizure focus (mass associated temporal lobe epilepsy, MaTLE; and paradoxical temporal lobe epilepsy, PTLE) DYN-like immunoreactivity was localized in the dentate granule cells and their mossy fiber terminals within the hilus and area CA3. In hippocampi that were the seizure focus (MTLE), 89% showed an additional band of immunoreactivity confined to the inner molecular layer (IML) of the dentate gyrus, representing recurrent mossy fiber collaterals. In 11% of MTLE patients no staining was found in the IML (MTLE/DYN-). The MTLE/DYN- hippocampi were also characterized by a significantly lower degree of cell loss than in MTLE hippocampi in the dentate granule cell layer, the hilus and CA3. Both MTLE and MTLE/DYN- hippocampi showed evoked epileptiform bursting in granule cells while MTLE showed greater polysynaptic EPSPs and spontaneous excitatory activity. Thus granule cell recurrent collateral sprouting may account for only some aspects of hyperexcitability. In 30% of the MTLE group, hilar neurons of a variety of morphological types expressed DYN immunoreactivity in their somata and dendrites. The density of [3H]U69,593 binding sites in MaTLE and PTLE patients was highest in areas CA1 and the subiculum-regions having little or no DYN-staining. In the dentate molecular layer, hilus and CA3--regions with the most DYN immunoreactivity--there was a low density of ligand binding. The significance of this transmitter/receptor mismatch is yet unknown.

Quantitative autoradiographic analysis of ionotropic glutamate receptor subtypes in human temporal lobe epilepsy: up-regulation in reorganized epileptogenic hippocampus.

Medically intractable temporal lobe epilepsy is a common disease typically associated with hippocampal damage (sclerosis) and synaptic remodelling. These changes could include increased glutamate receptor expression, enhancing excitability and the potential for neuronal injury. We directly assessed this hypothesis using quantitative in vitro receptor autoradiography to determine the densities of glutamate-, NMDA-, quisqualate/alpha-amino-3-hydroxy-5-methyl-isoxazoleproprionic acid (AMPA)- and kainic acid-preferring binding sites in surgically removed hippocampi from patients with mesial temporal lobe epilepsy (sclerosis; MTLE) and patients with mass-associated temporal lobe epilepsy (no sclerosis; MaTLE), compared with autopsy material. Neuronal cell counts and in situ total protein densities were also obtained. In general, MaTLE and autopsy binding densities were indistinguishable. In contrast, some regions of MTLE hippocampi exhibited decreased receptor densities, with a corresponding loss of protein. In the hilus and CA1, however, ligand binding densities did not differ from the comparison groups in spite of markedly reduced protein content, consistent with increased glutamate receptor density. Kainate-preferring sites were distributed differently from the other glutamate subtypes and were uniformly decreased throughout the MTLE hippocampus, except for a unique expression within the outer dentate molecular layer. Along with increased NMDA and AMPA receptor densities in the hilus and CA1, this distinctive population of kainate receptors establishes that increased glutamate receptor expression is a feature of the remodelled MTLE hippocampus. These observations suggest that enhanced sensitivity to glutamate may be an important element in the pathophysiology of temporal lobe epilepsy.

Parathyroid hormone-related peptide is produced by cultured cerebellar granule cells in response to L-type voltage-sensitive Ca2+ channel flux via a Ca2+/calmodulin-dependent kinase pathway.

Parathyroid hormone (PTH)-related peptide (PTHrP) is expressed in the adult mammalian brain, but its function is unknown. Here we show that PTHrP and the PTH/PTHrP receptor are products of cerebellar granule cells in primary culture. Granule cells maintained under depolarizing conditions (25 mM K+) make and release PTHrP. Further, PTHrP-(1-36) stimulates cAMP accumulation in granule neurons in a dose-dependent manner with half-maximal activation at approximately 16 nM. Granule cell PTHrP mRNA is activity-dependent, and the pathway of regulation depends absolutely on the flux of Ca2+ ions through the L-type voltage-sensitive Ca2+ channel and the Ca2+/calmodulin kinase cascade. PTHrP is therefore a neuropeptide whose regulation depends upon L-type voltage-sensitive Ca2+ channel activity, and the gene is expressed under conditions that promote granule cell survival.

Vasoactive intestinal polypeptide and its receptor changes in human temporal lobe epilepsy.

The distribution of the VIP receptor in the human hippocampus was studied by receptor autoradiography using [3-iodotyrosyl-125I]Vasoactive Intestinal Peptide (VIP) as a ligand, and the relationship of receptor distribution to the distribution of the peptide (visualized by immunocytochemistry) was examined in hippocampi surgically removed from patients with medically intractable temporal lobe epilepsy (TLE) and hippocampi obtained at autopsy from neurologically normal subjects. In the autopsy hippocampi and hippocampi from TLE patients with extrahippocampal temporal lobe lesions [125I]VIP binding was highest in the dentate molecular layer, with lower levels in the fields of Ammon's Horn (CA fields) and the subiculum. In hippocampi from patients with no temporal lobe lesions but considerable hippocampal neuronal loss there were significant elevations in the levels of ligand binding in all CA fields and the subiculum. Ligand binding densities in all CA fields of the patient hippocampi were strongly negatively correlated with neuronal numbers. Immunocytochemical localization of VIP shows no obvious change in the distribution patters of VIP immunoreactivity in the patient groups. This is the first demonstration of VIP and its receptor distribution in the human hippocampus. It is suggested that the elevated levels of receptor binding in the hippocampal seizure focus may indicate a mechanism for greater excitability of neurons and/or for their survivability in the face of the increased excitation and potential for injury in a seizure focus.

The cardiac glycoside ouabain potentiates excitotoxic injury of adult neurons in rat hippocampus.

We demonstrate that the enzyme family responsible for the restoration of the transmembrane cation balance, namely the sodium pump (Na+, K(+)-ATPase), plays a critical role in whether glutamate injures adult neurons in vivo. Partial inhibition of the sodium pump by the cardiac glycoside ouabain in young adult rats is not itself damaging. This treatment, however, markedly potentiates ordinarily subtoxic dosages of the glutamate analog kainic acid to produce limbic seizures and widespread neurodegeneration within the hippocampus in a pattern closely resembling that observed for human temporal lobe epilepsy.

Regional distributions of hippocampal Na+,K(+)-ATPase, cytochrome oxidase, and total protein in temporal lobe epilepsy.

Na+,K(+)-ATPase (the sodium pump) is a ubiquitous enzyme that consumes ATP to maintain an adequate neuronal transmembrane electrical potential necessary for brain function and to dissipate ionic transients. Reductions in sodium pump function augment the sensitivity of neurons to glutamate, increasing excitability and neuronal damage in vitro. Temporal lobe epilepsy (TLE) is one disease characterized by hyperexcitability and marked hippocampal neuronal losses that could depend in part, on impaired sodium pump capacity secondary to changes in sodium pump levels and/or insufficient ATP supply. To assess whether abnormalities in the sodium pump occur in this disease, we used [3H]ouabain to determine the density of Na+,K(+)-ATPase for each anatomic region of hippocampus by in vitro autoradiography. Tissues were surgically obtained from epileptic patients with hippocampal sclerosis and compared with specimens from patients with seizures originating from temporal lobe tumors and autopsy controls. Changes in cellular population arising from neuronal losses or gliosis were assessed by protein densities derived from quantitative computerized densitometry of Coomassie-stained tissue sections. We estimated regional differences in capacity for ATP generation by determining cytochrome c oxidase (CO) activity. Principal neurons of hippocampus exhibit high levels of sodium pump enzyme. Both epilepsy groups exhibited slight but significant increases in sodium pump density/unit mass of protein in the dentate molecular layer, CA2, and subiculum as compared with autopsy controls. Greater hilar sodium pump density was also observed in sclerotic hippocampi. In contrast, CO activity was reduced in both epilepsy types throughout hippocampus. Results suggest that although sodium pump protein in surviving neurons appears to be upregulated in epilepsy, sodium pump capacity may be limited by the reduced levels of CO activity. Functional reduction in sodium pump capacity may be an important factor in hyperexcitability and neuronal death.

Marginally elevated prolactin levels require magnetic resonance imaging and evaluation for acromegaly.

Marginal elevations in serum PRL concentration represent a particularly difficult diagnostic dilemma. In most cases, mild hyperprolactinemia is not associated with organic disease. Patients with menstrual disturbances, galactorrhea, and confirmed elevations in serum PRL should have a screening TSH to rule out primary hypothyroidism (5). In cases where there is no clear etiology of hyperprolactinemia, an MRI should be performed. Magnetic resonance imaging with gadolinium is more sensitive and specific than CT scanning in detecting all types of pituitary tumors and is the study of choice (4). Further, a serum IGF-1 level (or OGTT) should be obtained when clinical symptoms and/or a pituitary mass suggest the possibility of acromegaly. An individual with abnormal GH screening tests but an unremarkable MRI would be subjected to an especially careful follow-up, including IGF-1 and PRL levels every 6 to 12 months. In this way, early tumor growth may be detected making a surgical cure more likely (Fig. 1). Although we have stressed the importance of GH-producing tumors as a cause of hyperprolactinemia, other tumor types of the pituitary may do so as well. Most of these will be detected by MRI.

Ventromedial hypothalamic lesions in rats suppress counterregulatory responses to hypoglycemia.

The central nervous system has been implicated in the activation of counterregulatory hormone release during hypoglycemia. However, the precise loci involved are not established. To determine the role of the ventromedial hypoglycemia, we performed hypoglycemic clamp studies in conscious Sprague-Dawley rats with bilateral VMH lesions produced by local ibotenic acid injection 2 wk earlier. Rats with lesions in the lateral hypothalamic area, frontal lobe, sham operated (stereotaxic needle placement into hypothalamus without injection), and naive animals served as control groups. The clamp study had two phases. For the first hour plasma glucose was fixed by a variable glucose infusion at euglycemia (approximately 5.9 mM). Thereafter, for an additional 90 min, glucose was either allowed to fall to (a) mild hypoglycemia (approximately 3.0 mM) or (b) more severe hypoglycemia (approximately 2.5 mM). Glucagon and catecholamine responses of lateral hypothalamic area-, frontal lobe-lesioned, sham operated, and naive animals were virtually identical at each hypoglycemic plateau. In contrast, glucagon, epinephrine, and norepinephrine responses in the VMH-lesioned rats were markedly inhibited; hormones were diminished by 50-60% during mild and by 75-80% during severe hypoglycemia as compared with the other groups. We conclude that the VMH plays a crucial role in triggering the release of glucagon and catecholamines during hypoglycemia.

Glutamate up-regulates alpha 1 and alpha 2 subunits of the sodium pump in astrocytes of mixed telencephalic cultures but not in pure astrocyte cultures.

Prior work employing an in vitro model of the cerebral cortex has shown that sodium pump activity is a critical determinant for neuronal survival of glutamate stimulation. We have hypothesized that up-regulation of total brain sodium pump activity will protect against potential excitotoxins. Increased sodium pump activity could theoretically occur by changes in the reaction rate (short-term) and/or by increased levels of sodium pump protein (long-term) and is potentially complex since the three catalytic (a) subunit isoforms of the sodium pump are distributed in a highly variable, cell-specific pattern in the brain. Short-term regulation (seconds to minutes) has been well studied: brain sodium pump exhibits a large dynamic range. In contrast, the possibility of long-term modulation of sodium pump activity has not been extensively explored. We used isoform specific antibodies and [3H]ouabain binding to determine whether prolonged stimulation of sodium pump activity in rodent telencephalic cultures increased total sodium pump enzyme. Exposure of mixed neuronal-glial cultures to high levels of glutamate (10 mM) for 18 h, which is highly toxic to neurons, was associated with an approximately 80% increase in alpha 1 and alpha 2 subunit expression by glia. Induction of alpha 2 subunit immunoreactivity was also associated with comparable changes in [3H]ouabain binding, suggesting that the up-regulation corresponded to functional alpha 2 protein. Shorter (30 min) glutamate treatments, which also killed neurons, did not produce similar changes in sodium pump expression. In contrast to mixed cultures, pure astrocyte cultures had undetectable alpha 2 and alpha 3 and moderate levels of alpha 1 protein, as confirmed by low levels of [3H]ouabain binding. Glutamate treatment using this protocol was associated with a decrease in alpha 1 sodium pump expression. We conclude that long-term regulation of the sodium pump can be demonstrated in glia which have developed in the presence of neurons. Both alpha 1 and alpha 2 isoforms of the sodium pump are involved in this response to glutamate.

Cell-type specific expression of Na+, K(+)-ATPase catalytic subunits in cultured neurons and glia: evidence for polarized distribution in neurons.

Na+,K(+)-ATPase (the sodium pump) is a family of proteins consisting of catalytic (alpha) and glycoprotein (beta) subunit isoforms which are differentially expressed in excitable tissue. To gain insight into the cell-type distribution of sodium pump protein, we determined the expression pattern of fetal rat telencephalic cultures, of telencephalic cultures depleted of neurons, and of pure astrocyte cultures. Isoform-specific antibodies were used for immunoblotting and immunohistochemistry, with supplemental [3H]ouabain binding to assess levels of functional alpha 2/alpha 3 protein. The results show that neurons of mixed telencephalic cultures uniquely express alpha 3 and high levels of alpha 1. The marked similarity in the distribution of microtubule-associated protein-2 and alpha 1 immunocytochemical staining strongly suggests that alpha 1 subunits are enriched in dendrites. Further, highly correlative growth cone-associated protein-43 and alpha 3 staining is consistent with a preferential expression of alpha 3 subunits in axons, which are also characterized by low levels of alpha 1 and no alpha 2 immunoreactivity. Process-bearing glia are intimately associated with neuronal aggregates and express high levels of both alpha 1 and alpha 2 protein, as well as GFAP. Interestingly, polygonal, flat glia not within neuronal aggregates are weakly immunopositive only for alpha 1 and GFAP. Pure astrocytic cultures possess appreciable alpha 1 protein and GFAP, but lack both alpha 2 and alpha 3 immunoreactivity. As predicted by the immunohistochemical findings, [3H]ouabain binding was low in pure astrocytic cultures, and much higher in the neuron-enriched mixed cultures. These observations confirm that neurons express all three catalytic isoforms of the sodium pump. They also suggest that specific alpha-isoforms may be polarized to targeted membrane regions of neurons. Further, glia intimately associated with neurons express alpha 2, bind significant amounts of [3H]ouabain, and possess much higher levels of alpha 1 and GFAP compared to glia not near neurons. Thus, neurons may regulate glial sodium pump expression.

Inhibition of alpha 2/alpha 3 sodium pump isoforms potentiates glutamate neurotoxicity.

Excessive stimulation of neurons by glutamic acid initiates a destructive cascade of ion fluxes, cellular swelling, and death. Homeostatic mechanisms which rectify these disturbances depend largely upon transmembrane ion gradients maintained by Na+,K(+)-ATPase (NaP). We proposed that the neurotoxicity of glutamate is enhanced when the NaP capacity is exceeded, and therefore, that the degree of neuronal death varies inversely with endogenous NaP activity. To test this concept, we directly reduced NaP activity in cultured rat telencephalic cells using either the specific inhibitor ouabain, or dcAMP, and assessed whether these treatments increased glutamate-induced neuronal death. Since rodent NaP catalytic subunits possess both low (alpha 1) and high (alpha 2/alpha 3) affinity for ouabain, we were able to inhibit selectively the alpha 2 (principally glial) and alpha 3 (neuronal) catalytic subunits without affecting the alpha 1 isoform. Brief exposures (5-60 min) to high ouabain concentrations (1-10 mM), which blocks the activity of all three catalytic subunits, killed differentiated neurons but spared glia. In contrast, differential inhibition of the alpha 2/alpha 3 isoforms (by 1 microM ouabain) was not of itself toxic, but produced a supersensitivity to glutamate. [3H]Ouabain binding studies confirmed that the glutamate neurotoxicity observed varied inversely with the degree of NaP inhibition. Further, this relationship was not absolutely dependent upon ouabain, since reductions in alpha 2/alpha 3 pump activity induced by dcAMP also amplified glutamate toxicity. We conclude that inhibition of neuronal NaP with high affinity for ouabain is not lethal to unstimulated cells, but markedly increases susceptibility to glutamate excitotoxicity.(ABSTRACT TRUNCATED AT 250 WORDS)

The A2 isoform of rat Na+,K(+)-adenosine triphosphatase is active and exhibits high ouabain affinity when expressed in transfected fibroblasts.

The alpha isoforms of the Na+,K(+)-ATPase (Na+ pump) are expressed with developmental and tissue heterogeneity in rodents and possess different sensitivity to inhibition by ouabain. We directly characterized the ouabain sensitivity of the rat A2 (alpha 2) isoform by transfecting NIH 3T3 cells with rat A2. The treated cells exhibit high affinity (40 nM) ouabain binding with a density of 2 pmol/mg protein. 86Rb+ flux studies confirm that A2 is functional in this system and that A2 is inhibited by submicromolar concentrations of ouabain. These findings are consistent with measurements of ouabain affinity in tissues which express the A2 isoform.