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T lymphocytes are exposed to hypoxia during their development and also when they migrate to hypoxic pathological sites such as tumors and wounds. Although hypoxia can affect T cell development and function, the mechanisms by which immune cells sense and respond to changes in O(2)-availability are poorly understood. K(+) channels encoded by the Kv1.3 subtype of the voltage-dependent Kv1 gene family are highly expressed in lymphocytes and are involved in the control of membrane potential and cell function. In this study, we investigate the sensitivity of Kv1.3 channels to hypoxia in freshly isolated human T lymphocytes and leukemic Jurkat T cells. Acute exposure to hypoxia (20 mmHg, 2 min) inhibits Kv1.3 currents in both cell types by 20%. Prolonged exposure to hypoxia (1% O(2) for 24 h) selectively decreases Kv1.3 protein levels in Jurkat T cells by 47%, but not Kvbeta2 and SK2 Ca-activated K(+) channel subunit levels. The decrease in Kv1.3 protein levels occurs with no change in Kv1.3 mRNA expression and is associated with a significant decrease in K(+) current density. A decrease in Kv1.3 polypeptide levels similar to that obtained during hypoxia is produced by Kv1.3 channel blockage. Our results indicate that hypoxia produces acute and long-term inhibition of Kv1.3 channels in T lymphocytes. This effect could account for the inhibition of lymphocyte proliferation during hypoxia. Indeed, we herein present evidence showing that hypoxia selectively inhibits TCR-mediated proliferation and that this inhibition is associated with a decrease in Kv1.3 proteins.

The purpose of the present studies was to examine the renal distribution and functional properties of Na(+)-HCO(3)(-) cotransporter type 4 (NBC4), the latest NBC isoform to be identified. Zonal distribution studies in rat kidney by Northern blot hybridization and RT-PCR demonstrated that NBC4 is highly abundant in the outer medulla and cortex but is low in the inner medulla. Nephron segment distribution studies indicated that NBC4 is predominantly expressed in the medullary and cortical thick ascending limb of the loop of Henle. Using specific primers on the basis of the published sequence (GenBank accession no. AF-207661), a full-length NBC4 variant was cloned from human liver and examined. The sequence of this variant (called NBC4e) is shorter by 86 amino acids vs. the published sequence. Xenopus laevis oocytes injected with the full-length NBC4e cRNA were compared with NBC1-expressing oocytes. Although exposure of NBC1-expressing oocytes to CO(2)/HCO(3)(-) resulted in immediate hyperpolarization, the NBC4-expressing oocytes did not show any alteration in membrane potential. NBC activity in oocytes, assayed as the Na(+)-dependent, HCO(3)(-)-mediated intracellular pH recovery from acidosis, indicated that NBC4 is a DIDS-inhibitable NBC. We propose that NBC4 is expressed in the thick ascending limb of the loop of Henle and mediates cellular HCO(3)(-) uptake in this segment.

Rett syndrome is a progressive neurodevelopmental disorder with a well-defined clinical spectrum and course. Recently, mutations in the gene encoding X-linked methyl-CpG binding protein 2 (MECP2) have been identified as the cause of Rett syndrome. Along with the classic form, variant forms of Rett syndrome and Rett syndrome phenotypes are also recognized. We report on a girl who, at age 2 months, developed an acute encephalopathy with destructive brain damage 12 hours after acellular pertussis vaccination. Peripheral lymphocyte subset analysis revealed the existence of T lymphocytes double positive for CD4 and CD8 markers. This pattern normalized over the following 3 months. Months later, the girl manifested a Rett syndrome phenotype. DNA screening of the MECP2 gene was unrevealing in the child and her parents. This previously unreported association emphasizes the notion that Rett syndrome phenotypes can result from different (either genetic or environmental) causes.

The Ca(2+)-binding proteins KChIP1-4 (KChIP3 is also known as DREAM and calsenilin) act as auxiliary subunits for voltage-gated K(+) channels in the Kv4 family. Here we identify three splicing isoforms of rat KChIP2 with variable N-terminal peptides. The two longer isoforms, which contain the 32-amino acid peptide, produce larger increases in Kv4.3 protein level and current density and more effectively localize themselves and their associated channels at the plasma membrane than the shortest variant. The 32-amino acid peptide contains potential palmitoylation cysteines. Metabolic labeling demonstrates that these cysteines in the KChIP2 isoforms, as well as the corresponding sites in KChIP3, are palmitoylated. Mutating these cysteines reduces their plasma membrane localization and the enhancement of Kv4.3 current density. Thus, palmitoylation of the KChIP auxiliary subunits controls plasma membrane localization of their associated channels.

We (Thorne GD, Shimizu S, and Paul RJ. Am J Physiol Cell Physiol 281: C24-C32, 2001) have recently shown that organ culture for 24 h specifically inhibits relaxation to acute hypoxia (95% N(2)-5% CO(2)) in the porcine coronary artery. Here we show similar results in the porcine carotid artery and the rat and mouse aorta. In the coronary artery, part of the inability to relax to hypoxia after organ culture is associated with a concomitant loss in ability to reduce intracellular Ca(2+) concentration ([Ca(2+)](i)) during hypoxia (Thorne GD, Shimizu S, and Paul RJ. Am J Physiol Cell Physiol 281: C24-C32, 2001). To elucidate the mechanisms responsible for the loss of relaxation to hypoxia, we investigated changes in K(+) and Ca(2+) channel activity and gene expression that play key roles in [Ca(2+)](i) regulation in vascular smooth muscle (VSM). Reduced mRNA expression of O(2)-sensitive K(+) channels (Kv1.5 and Kv2.1) was shown by reverse transcriptase-polymerase chain reaction in the rat aorta. In contrast, no change in other expressed voltage-gated K(+) channels (Kv1.2 and Kv1.3) or Ca(2+) channel subtypes was found. Modified K(+) channel expression is supported by functional evidence indicating a reduced response to general K(+) channel activation, by pinacidil, and to specific voltage-dependent K(+) (Kv) channel blockade by 4-aminopyridine. In conclusion, organ culture decreases expression of specific Kv channels. These changes are consistent with altered mechanisms of VSM contractility that may be involved in Ca(2+)-dependent pathways of hypoxia-induced vasodilation.

The slow Wallerian degeneration mouse (C57BL/Wld(s)) is a mutant strain of mouse, with the unique phenotype of prolonged survival of the distal axon following axotomy. The causative mutation is an 85 kb tandem triplication on distal mouse chromosome 4. The dominant slow Wallerian degeneration phenotype is conferred by a hybrid gene within the triplication, comprising a gene of previously unknown function, D4Cole1e, and the 5' end of ubiquitination factor E4B (Ube4b). It encodes an in-frame fusion protein consisting of the N-terminal 70 amino acids of Ube4b and 303 amino acids derived from the D4Cole1e gene. We have identified the human homologue of D4Cole1e, and mapped it to chromosome 1p36.2. Additional fluorescence in situ hybridisation signals indicate the presence of several homologous human sequences. Northern blot analysis shows two transcripts, widely expressed at varying levels in different human tissues. The human cDNA, which encodes a protein of 279 amino acids, has 80% nucleotide identity with the mouse cDNA. The derived human and mouse protein sequences share 78% amino acid identity and 82% amino acid similarity. The human cDNA and protein sequences are identical to the human nicotinamide mononucleotide adenylyltransferase (NMNAT). We have also determined the intron/exon structure of the gene, which will facilitate the screening of these exons for mutations in human neurodegenerative disorders.

Axons and their synapses distal to an injury undergo rapid Wallerian degeneration, but axons in the C57BL/WldS mouse are protected. The degenerative and protective mechanisms are unknown. We identified the protective gene, which encodes an N-terminal fragment of ubiquitination factor E4B (Ube4b) fused to nicotinamide mononucleotide adenylyltransferase (Nmnat), and showed that it confers a dose-dependent block of Wallerian degeneration. Transected distal axons survived for two weeks, and neuromuscular junctions were also protected. Surprisingly, the Wld protein was located predominantly in the nucleus, indicating an indirect protective mechanism. Nmnat enzyme activity, but not NAD+ content, was increased fourfold in WldS tissues. Thus, axon protection is likely to be mediated by altered ubiquitination or pyridine nucleotide metabolism.

Background: Na+:HCO3- cotransporters mediate the transport of HCO3- into or out of the cell. We recently reported the partial cloning and characterization of a new human Na+:HCO3- cotransporter (referred to as NBC-3 or kNBC-3). The purpose of the present studies was to clone the mouse kNBC-3 and to examine its properties and expression in the kidney., Methods: Using primers from human kNBC-3 cDNA and 5' and 3' rapid amplification cDNA end polymerase chain reaction (RACE PCR), the mouse kNBC-3 full-length cDNA was cloned from inner medullary collecting duct (mIMCD-3) cells. The tissue distribution and functional properties of NBC-3 was determined using established methods., Results: The coding region of the mouse kNBC-3 has 1089 amino acids and shows 73 and 56% identity to human NBC-2 and NBC-1, respectively. The renal distribution of kNBC-3 demonstrated a unique expression pattern: Whereas kNBC-1 is predominantly expressed in the cortex and is absent in the inner medulla, kNBC-3 shows an intense expression level in the inner medulla and is absent in the cortex. Expression studies in oocytes indicated that NBC-3 mediates Na-dependent HCO3- cotransport. Electrophysiological experiments demonstrated that unlike kNBC-1, which is electrogenic, kNBC-3 is electroneutral., Conclusions: Based on its distribution and electroneutrality, we propose that kNBC-3 mediates the transport of HCO3- into the cells.

Exons of three genes were identified within the 85-kilobase tandem triplication unit of the slow Wallerian degeneration mutant mouse, C57BL/Wld(S). Ubiquitin fusion degradation protein 2 (Ufd2) and a previously undescribed gene, D4Cole1e, span the proximal and distal boundaries of the repeat unit, respectively. They have the same chromosomal orientation and form a chimeric gene when brought together at the boundaries between adjacent repeat units in Wld(S). The chimeric mRNA is abundantly expressed in the nervous system and encodes an in-frame fusion protein consisting of the N-terminal 70 amino acids of Ufd2, the C-terminal 302 amino acids of D4Cole1e, and an aspartic acid formed at the junction. Antisera raised against synthetic peptides detect the expected 43-kDa protein specifically in Wld(S) brain. This expression pattern, together with the previously established role of ubiquitination in axon degeneration, makes the chimeric gene a promising candidate for Wld. The third gene altered by the triplication, Rbp7, is a novel member of the cellular retinoid-binding protein family and is highly expressed in white adipose tissue and mammary gland. The whole gene lies within the repeat unit leading to overexpression of the normal transcript in Wld(S) mice. However, it is undetectable on Northern blots of Wld(S) brain and seems unlikely to be the Wld gene. These data reveal both a candidate gene for Wld and the potential of the Wld(S) mutant for studies of ubiquitin and retinoid metabolism.

The present study was undertaken to determine whether rat carotid bodies express adenosine (Ado) A(2A) receptors and whether this receptor is involved in the cellular response to hypoxia. Our results demonstrate that rat carotid bodies express the A(2A) and A(2B) Ado receptor mRNAs but not the A(1) or A(3) receptor mRNAs as determined by reverse transcriptase-polymerase chain reaction. In situ hybridization confirmed the expression of the A(2A) receptor mRNA. Immunohistochemical studies further showed that the A(2A) receptor is expressed in the carotid body and that it is colocalized with tyrosine hydroxylase in type I cells. Whole cell voltage-clamp studies using isolated type I cells showed that Ado inhibited the voltage-dependent Ca(2+) currents and that this inhibition was abolished by the selective A(2A) receptor antagonist ZM-241385. Ca(2+) imaging studies using fura 2 revealed that exposure to severe hypoxia induced elevation of intracellular Ca(2+) concentration ([Ca(2+)](i)) in type I cells and that extracellularly applied Ado significantly attenuated the hypoxia-induced elevation of [Ca(2+)](i). Taken together, our findings indicate that A(2A) receptors are present in type I cells and that activation of A(2A) receptors modulates Ca(2+) accumulation during hypoxia. This mechanism may play a role in regulating intracellular Ca(2+) homeostasis and cellular excitability during hypoxia.

One of the early events in O2 chemoreception is inhibition of O2-sensitive K+ (KO2) channels. Characterization of the molecular composition of the native KO2 channels in chemosensitive cells is important to understand the mechanism(s) that couple O2 to the KO2 channels. The rat phaeochromocytoma PC12 clonal cell line expresses an O2-sensitive voltage-dependent K+ channel similar to that recorded in other chemosensitive cells. Here we examine the possibility that the Kv1.2 alpha-subunit comprises the KO2 channel in PC12 cells. Whole-cell voltage-clamp experiments showed that the KO2 current in PC12 cells is inhibited by charybdotoxin, a blocker of Kv1.2 channels. PC12 cells express the Kv1.2 alpha-subunit of K+ channels: Western blot analysis with affinity-purified anti-Kv1.2 antibody revealed a band at approximately 80 kDa. Specificity of this antibody was established in Western blot and immunohystochemical studies. Anti-Kv1.2 antibody selectively blocked Kv1.2 current expressed in the Xenopus oocyte, but had no effect on Kv2.1 current. Anti-Kv1.2 antibody dialysed through the patch pipette completely blocked the KO2 current, while the anti-Kv2.1 and irrelevant antibodies had no effect. The O2 sensitivity of recombinant Kv1.2 and Kv2.1 channels was studied in Xenopus oocytes. Hypoxia inhibited the Kv1.2 current only. These findings show that the KO2 channel in PC12 cells belongs to the Kv1 subfamily of K+ channels and that the Kv1.2 alpha-subunit is important in conferring O2 sensitivity to this channel.

Little is known about the molecular composition of the O2-sensitive K+ (Ko2) channels. The possibility that these channels belong to the Shaker subfamily (Kv1) of voltage-dependent K+ (Kv) channels has been raised in pulmonary artery (PA) smooth muscle cells. Numerous findings suggest that the Ko2 channel in PC12 cells is a Kv1 channel, formed by the Kv1.2 alpha subunit. The Ko2 channel in PC12 cells is a slow-inactivating voltage-dependent K+ channel of 20 pS conductance. Other Kv channels, also expressed in PC12 cells, are not inhibited by hypoxia. Selective up-regulation by chronic hypoxia of the Kv1.2 alpha subunit expression correlates with an increase O2-sensitivity of the K+ current. Other Kv1 alpha subunit genes encoding slow-inactivating Kv channels, such as Kv1.3, Kv2.1, Kv3.1 and Kv3.2 are not modulated by chronic hypoxia. The Ko2 current in PC12 cells is blocked by 5 mM externally applied tetraethylammonium chloride (TEA) and by charydbotoxin (CTX). The responses of the Kv1.2 K+ channel to hypoxia have been studied in the Xenopus oocytes and compared to those of Kv2.1, also proposed as Ko2 channel in PA smooth muscle cells. Two-electrode voltage clamp experiments show that hypoxia induces inhibition of K+ current amplitude only in oocytes injected with Kv1.2 cRNA. These data indicate that Kv1.2 K+ channels are inhibited by hypoxia.

Kinesin and kinesin superfamily proteins are molecular motors involved in important intracellular functions such as organelle transport and cell division. They are microtubule-activated ATPases composed of a motor domain that binds to microtubules and a cargo-binding domain that binds to specific organelles. While searching for the slow Wallerian degeneration mutation (WldS) on distal mouse Chromosome (Chr) 4, we have identified a member of the kinesin superfamily whose predicted gene product has the N-terminal motor domain of Kif1b and a novel C-terminal cargo-binding domain homologous to Kif1a. Kif1b is responsible for the movement of mitochondria along the axon, but the novel isoform containing the alternative C-terminal domain is likely to have a different cargo-binding specificity. cDNA library screening and Northern blot analysis indicate that the alternatively spliced form of Kif1b containing the novel 3'end accounts for the most part of Kif1b expression. We also found more alternatively spliced exons that can give rise to heterogeneous transcripts. Therefore, alternative splicing, as well as multiple genes, may contribute to the selective movement of diverse organelles by anterograde axonal transport. Kif1b maps on distal mouse Chr 4, within the Wld genetic candidate interval, but outside the recently identified triplication. There is, however, no evidence that Kif1b is the Wld gene.

1. Adenosine has been shown to decrease Ca2+ current (ICa) and attenuate the hypoxia-induced enhancement of intracellular free Ca2+ ([Ca2+]i) in oxygen-sensitive rat phaeochromocytoma (PC12) cells. These effects are mediated via the adenosine A2A receptor and protein kinase A (PKA). The current study was undertaken to determine the effects of adenosine on Ca2+ current and hypoxia-induced change in [Ca2+]i during chronic hypoxia. 2. Whole cell patch-clamp studies revealed that the effect of adenosine on ICa was significantly reduced when PC12 cells were exposed to hypoxia (10 % O2) for 24 and 48 h. 3. Ca2+ imaging studies using fura-2 revealed that the anoxia-induced increase in [Ca2+]i was significantly enhanced when PC12 cells were exposed to 10 % O2 for up to 48 h. In contrast, the inhibitory effects of adenosine on anoxia-induced elevation of [Ca2+]i was significantly blunted in PC12 cells exposed to hypoxia for 48 h. 4. Northern blot analysis revealed that mRNA for the A2A receptor, which is the only adenosine receptor subtype expressed in PC12 cells, was significantly upregulated by hypoxia. Radioligand binding analysis with [3H]CGS21680, a selective A2A receptor ligand, showed that the number of adenosine A2A receptor binding sites was similarly increased during exposure to 10% O2 for 48 h. 5. PKA enzyme activity was significantly inhibited when PC12 cells were exposed to 10% O2 for 24 and 48 h. However, we found that hypoxia failed to induce change in adenosine- and forskolin-stimulated adenylate cyclase enzyme activity. Chronic hypoxia also did not alter the immunoreactivity level of the G protein Gsalpha, an effector of the A2 signalling pathway. 6. Whole cell patch-clamp analysis showed that the effect of 8-bromo-cAMP, an activator of PKA, on ICa was significantly attenuated during 48 h exposure to 10% O2.7. We conclude therefore that the reduced effect of adenosine on ICa and [Ca2+]i in PC12 cells exposed to chronic hypoxia is due to hypoxia-induced downregulation of PKA. This mechanism may serve to reduce the negative feedback on ICa and [Ca2+]i by adenosine and therefore maintain enhanced membrane excitability of PC12 cells during long-term hypoxia.

Wallerian degeneration is the degeneration of the distal stump of an injured axon. It normally occurs over a time course of around 24 hr but it is delayed in the slow Wallerian degeneration mutant mouse (C57BL/Wlds) for up to 3 weeks. The gene, which protects from rapid Wallerian degeneration, Wld, previously has been mapped to distal chromosome 4. This paper reports the fine genetic mapping of the Wld locus, the generation of a 1.4-Mb bacterial artificial chromosome and P1 artificial chromosome contig, and the identification of an 85-kb tandem triplication mapping within the candidate region. The mutation is unique to C57BL/Wlds among 36 strains tested and therefore is a strong candidate for the mutation that leads to delayed Wallerian degeneration. There are very few reports of tandem triplications in a vertebrate and no evidence for a mutation mechanism so this unusual mutation was characterized in more detail. Sequence analysis of the boundaries of the repeat unit revealed a minisatellite array at the distal boundary and a matching 8-bp sequence at the proximal boundary. This finding suggests that recombination between short homologous sequences ("illegitimate" or "nonhomologous" recombination) was involved in the rearrangement. In addition, a duplication allele was identified in two Wlds mice, indicating some instability in the repeat copy number and suggesting that the triplication arose from a duplication by unequal crossing over.

Lesions of the first motor neuron provoke abnormal voluntary movements. To clarify the central nervous system mechanisms underlying these changes we analyzed the behavior of the first motor unit recruited during a minimal effort tonic contraction of the deltoid and abductor digiti minimi manus muscles in patients with hemiplegia due to cerebrovascular lesions in the distribution of the middle cerebral artery. We compared data from paretic and healthy muscles in the same subject. The onset and recruitment intervals determined for the single motor unit yielded the range of control. The first recruited motor unit had a lower baseline firing rate and the second recruited motor unit potential appeared significantly earlier (p < 0.01) in plegic than in healthy muscles. Both changes affected distal more than proximal locations. Recordings from plegic muscles, particularly at distal locations, also disclosed a lowered range of control. These findings suggest that in hemiplegic patients the central nervous system loses its ability to modulate the frequency of firing during minimal effort voluntary movements so that distal muscles tend to behave like proximal muscles.

1. The present study was undertaken to determine the role of adenosine in mediating the cellular responses to hypoxia in rat phaeochromocytoma (PC12) cells, an oxygen-sensitive clonal cell line. 2. Reverse transcriptase polymerase chain reaction studies revealed that PC12 cells express adenosine deaminase (the first catalysing enzyme of adenosine degradation) and the A2A and A2B adenosine receptors, but not the A1 or A3 adenosine receptors. 3. Whole-cell current- and voltage-clamp experiments showed that adenosine attenuated the hypoxia-induced membrane depolarization. The hypoxia-induced suppression of the voltage-sensitive potassium current (IK(V)) was markedly reduced by adenosine. Furthermore, extracellularly applied adenosine increased the peak amplitudes of IK(V) in a concentration-dependent manner. This increase was blocked by pretreatment not only with a non-specific adenosine receptor antagonist, 8-phenyltheophylline (8-PT), but also with a selective A2A receptor antagonist, ZM241385. 4. Ca2+ imaging studies using fura-2 acetoxymethyl ester (fura-2 AM) revealed that the increase in intracellular free Ca2+ during hypoxic exposure was attenuated significantly by adenosine. Voltage-clamp studies showed that adenosine inhibited the voltage-dependent Ca2+ currents (ICa) in a concentration-dependent fashion. This inhibition was also abolished by both 8-PT and ZM241385. 5. The modulation of both IK(V) and ICa by adenosine was prevented by intracellular application of an inhibitor of protein kinase A (PKA), PKA inhibitor fragment (6-22) amide. In addition, the effect of adenosine on either IK(V) or ICa was absent in PKA-deficient PC12 cells. 6. These results indicate that the modulatory effects of adenosine on the hypoxia-induced membrane responses of PC12 cells are likely to be mediated via activation of the A2A receptor, and that the PKA pathway is required for these modulatory actions. We propose that this modulation serves to regulate membrane excitability in PC12 cells and possibly other oxygen-sensitive cells during hypoxia.

PC-12 cells depolarize during hypoxia and release dopamine. The hypoxia-induced depolarization is due to inhibition of an O2-sensitive K+ current. The role of dopamine released during hypoxia is uncertain, but it could act as an autocrine to modulate membrane conductance during hypoxia. The current study was undertaken to investigate this possibility. Reverse transcription-polymerase chain reaction and sequence analysis revealed that the D2 isoform of the dopamine receptor is expressed in rat PC-12 cells. Exogenously applied dopamine and the D2 agonist quinpirole elicited inhibition of a voltage-dependent K+ current (I(K)) that was prevented by sulpiride, a D2 receptor antagonist. Dopamine and quinpirole applied during hypoxia potentiated the inhibitory effect of hypoxia on I(K). We also found that quinpirole caused reversible inhibition of a voltage-dependent Ca2+ current (I(Ca)) and attenuation of the increase in intracellular free Ca2+ during hypoxia. Our results indicate that dopamine released from PC-12 cells during hypoxia acts via a D2 receptor to "autoregulate" I(K) and I(Ca).

1. Electrophysiological (single-channel patch clamp) and molecular biological experiments (reverse transcriptase-polymerase chain reaction) were performed to attempt to identify the O2-sensitive K+ channel in rat phaeochromocytoma (PC12) cells. 2. Four types of K+ channels were recorded in PC12 cells: a small-conductance K+ channel (14 pS), a calcium-activated K+ channel (KCa; 102 pS) and two K+ channels with similar conductance (20 pS). These last two channels differed in their time-dependent inactivation: one was a slow-inactivating channel, while the other belonged to the family of fast transient K+ channels. 3. The slow-inactivating 20 pS K+ channel was inhibited by hypoxia. Exposure to hypoxia produced a 50% reduction in channel activity (number of active channels in the patch x open probability). Hypoxia had no effect on the 20 pS transient K+ channels, whereas reduced O2 stimulated the KCa channels. 4. The genes encoding the alpha-subunits of slow-inactivating K+ channels for two members of the Shaker subfamily of K+ channels (Kv1.2 and Kv1.3) together with the Kv2.1, Kv3.1 and Kv3.2 channel genes were identified in PC12 cells. 5. The expression of the Shaker Kv1.2, but none of the other K+ channel genes, increased in cells exposed to prolonged hypoxia (18 h). The same cells were more responsive to a subsequent exposure to hypoxia (35% inhibition of K+ current measured in whole-cell voltage clamp) compared with the cells maintained in normoxia (19% inhibition). 6. These results indicate that the O2-sensitive K+ channel in PC12 cells is a 20 pS slow-inactivating K+ channel that is upregulated by hypoxia. This channel appears to belong to the Shaker subfamily of voltage-gated K+ channels.

In rat white adipocytes histamine is oxidized by a semicarbazide-sensitive amine oxidase which has benzylamine or preferential substrate (Bz-SSAO). To determine whether Bz-SSAO could control the extracellular levels of histamine and other histamine-related compounds active in lipid mobilization, a series of histaminergic compounds was screened as possible substrates or inhibitors of Bz-SSAO activity. Histaminergic compounds with imidazolo or thiazolo groups are oxidized by rat white-adipocyte Bz-SSAO whereas S-isothiourea derivatives, with two- or three-carbon-atom alkyl chains between the isothiourea and the N,N-dimethyl residue are, instead, inhibitors of the enzyme. Amtamine has been identified as a selective, high affinity substrate for rat white adipocyte Bz-SSAO. This enzymatic degradation might represent a catabolic pathway for the drug. These results show that the histaminase property of the rat white-adipocyte enzyme Bz-SSAO also extends to other histamine derivatives active at histamine receptors.

Carotid body type I cells and the O2 sensitive pheochromocytoma (PC12) cells release dopamine during hypoxia. Reduced O2 tension causes inhibition of an outward rectifying the O2-sensitive potassium (K) channel in the O2-sensitive pheochromocytoma (PC12) cell line, which leads to membrane depolarization and increased intracellular free Ca2+. We found that removal of Ca2+ from the extracellular milieu, inhibition of voltage-dependent Ca2+ channels, and chelation of intracellular Ca2+ prevents full activation of the TH gene expression during hypoxia. These findings suggest that membrane depolarization and regulation of intracellular free Ca2+ are critical signal transduction events that regulate expression of the TH gene in PC12 cells during hypoxia. Gene expression of tyrosine hydroxylase (TH), the rate-limiting enzyme in the biosynthesis of dopamine, is stimulated by reduced O2 tension in both type I cells and PC12 cells. The increase in TH gene expression in PC12 cells during hypoxia is due to increases in both the rate of transcription and mRNA stability. Analysis of reporter-gene constructs revealed that increased transcription of the TH gene during hypoxia is regulated by a region of the proximal promoter that extends from -284 to -150 bases, relative to the transcription start site. This region of the gene contains a number of cis-acting regulatory elements including AP1, AP2 and hypoxia-inducible factor (HIF-1). Competition assays revealed that hypoxia-induced binding occurs at both the AP1 and HIF-1 sites. Results from super-shift and shift Western assays showed that a heterodimer consisting of c-Fos and JunB binds to the AP1 site during hypoxia. Mutagenesis experiments revealed that the AP1 site is required for increased transcription of the TH gene during hypoxia. We also found that the genes that encode the c-Fos and JunB transcription factor proteins are regulated by reduced O2 tension.

We have shown that the PC12 cell line is an excellent model system for investigations of the molecular and cellular processes involved in O2-chemosensitivity. We have identified an O2-sensitive K channel in this cell line that mediates membrane depolarization, an increase in intracellular free Ca2+, and dopamine release during hypoxia. We also presented evidence which shows that expression of the gene for tyrosine hydroxylase, the rate-limiting enzyme in dopamine biosynthesis, is stimulated by reduced O2 tension in PC12 and type I carotid body cells. In addition, we have successfully identified the DNA sequences and trans-acting protein factors that regulate transcription of the TH gene during hypoxia. The mechanisms by which a reduction in O2 tension is transduced into alter cell function including increased gene expression remain unknown. Unpublished results from our laboratory show that the increased TH gene expression during hypoxia does not require activation of the cAMP-PKA signal transduction pathway. We propose that the increase in intracellular free Ca2+ that occurs as a result of membrane depolarization might play an important role. Preliminary findings from our laboratory show that blockade of the voltage operated Ca2+ channel or chelation of intracellular Ca2+ prevent full activation of the TH gene during hypoxia.

Diacylglycerol analogs and phorbol esters are used as protein kinase C (PKC) activators to investigate the effect of PKC on L-type calcium current [ICa(L)] in cardiomyocytes. 1-2-dioctanoyl-sn-glycerol (diC8) is a potent analog of diacylglycerol (DAG) which produces positive inotropic effects in guinea-pig atria cardiomyocytes via PKC activation. DiC8 effect on ICa(L), recorded (at 25 degrees C) in whole-cell voltage-clamp, was measured in 17-day-old embryonic chick cardiomyocytes in culture. ICa(L) was recorded in Na+, K(+)-free solution (external) and Ca(2+)-, K(+)-free solution (pipette), with depolarizing steps (to +10 mV) applied from a holding potential of -40 mV. Perfusion with different concentrations of diC8 (from 0.1 to 100 microM) inhibited ICa(L) in a dose-dependent manner, with half-maximal inhibition occurring at 12.5 microM. The effect of diC8 occurred rapidly, the effect beginning within 2 min and being completed within 5 min. In order to determine if the inhibitory effect of diC8 on ICa(L) was through activation of PKC, 25 microM diC8 was applied after pre-incubation of the cardiomyocytes with the PK inhibitors staurosporine (1 microM) or H-7 (50 microM). The effect of diC8 was not prevented by staurosporine or H-7. To further rule out the involvement of PKC in the action of diC8, experiments were performed using another analog of DAG, 1-oleyl-2-acetyl-glycerol (OAG, 50 microM) and Angiotensin-II (A-II, 0.1 microM). OAG failed to produce any effect on ICa(L). A-II, believed to act by activation of PKC did not affect ICa(L) within a test period of 8 min.(ABSTRACT TRUNCATED AT 250 WORDS)

The objective was to determine if the benzothiazepine compound CGP-37157 selectively inhibits the Na(+)-Ca2+ exchanger of cardiac mitochondria without affecting the L-type voltage-dependent calcium channel, the Na(+)-Ca2+ exchanger, or the Na(+)-K(+)-ATPase of the cardiac sarcolemma, or the Ca(2+)-ATPase of the cardiac sarcoplasmic reticulum. Mitochondrial Na(+)-Ca2+ exchange activity was determined by monitoring intramitochondrial free [Ca2+] in isolated heart mitochondria loaded with the Ca(2+)-sensitive fluorophore fura-2. CGP-37157 inhibited the activity of mitochondrial Na(+)-Ca2+ exchange in a dose-dependent manner (IC50 0.36 microM). Calcium currents were recorded by whole-cell voltage clamp in isolated neonatal ventricular myocytes. Diltiazem was able to block the recorded current completely, thus confirming the current to be exclusively L-type. CGP-37157 had no effect on the calcium current recorded under identical conditions. CGP-37157, at concentrations < or = 10 microM, had no effect on the activities of the Na(+)-Ca2+ exchanger and Na(+)-K(+)-ATPase in isolated cardiac sarcolemmal vesicles or on activity of the Ca(2+)-ATPase in isolated cardiac sarcoplasmic reticulum vesicles. The data suggest that CGP-37157 is a potent, selective, and specific inhibitor of mitochondrial Na(+)-Ca2+ exchange at concentrations < or = 10 microM.

In cardiomyocytes of some animals, the Na+ current shows slow gating properties, and seems to contribute to the plateau phase of the action potential. In the present study, we characterized the fast Na+ current in freshly-isolated ventricular cardiomyocytes from 18-day-old fetal rats, using whole-cell patch clamp, and examined a possible contribution of the Na+ current to action potential duration (APD). Currents were recorded (at 25 degrees C) in Ca(2+)-K(+)-free external and internal (pipette) solutions with 143 mM [Na]o and 20 mM [Na]i. The fast Na+ current was elicited by depolarizing steps above -47 mV from a holding potential of -97 mV. The maximal amplitude of the current was 735 +/- 94 pA (mean +/- S.E., n = 11) at -7 mV. The current density was 39.0 +/- 6.7 pA/pF (n = 9). The reversal potential was 51 +/- 3 mV (n = 11), agreeing with the equilibrium potential for Na+ (51 mV). The inward current was completely blocked in absence of [Na]o and by tetrodotoxin (TTX, 10 microns). The inactivation curve (h infinity) was sigmoidal between -127 and -27 mV, the half-inactivation potential and slope factor (k) being -69 and 9.5 mV, respectively. The activation curve (normalized gNa) was also sigmoidal, with half-activation voltage and k of -27 mV and -8.4 mV, respectively. The two curves overlapped from -60 to -30 mV. The current decay was best fitted by a two-exponential equation: (a) the time constant of the fast component (tau if) decreased from 3.9 +/- 0.6 ms at -37 mV to 0.8 +/- 0.1 ms at 23 mV; (b) the slow component (tau is) declined from 31 +/- 8 ms to 16 +/- ms at the same potentials. The recovery from inactivation had also two components: fast (tau rf = 10 +/- 2 ms) and slow (tau rs = 307 +/- 44 ms). TTX (1 micron) decreased the slow inactivating component and ADP50 by 69.5 +/- 4.2% and 25 +/- 6%, respectively. Therefore, the Na+ current in rat fetal cardiomyocytes may significantly contribute to the plateau phase of the action potential. The kinetics properties of the fast Na+ current of the 18-day fetal cardiomyocytes were compared with those from 1-day old neonatal rats. The current density was lower and the current decay was slower in the fetal cells in comparison with neonatal cells.

The effects of the Ca2+ channel agonist, Bay K 8644, on the slow (L-type) Ca2+ channels was examined in young (3-day-old) embryonic chick heart cells, which naturally exhibit long-lasting openings. Bay K 8644 (5 microM) increased (a) the peak amplitude of the ensemble-averaged current by 3.9 +/- 0.9-fold (mean +/- S.E.) and (b) the maximal number of simultaneous opening from 2.6 +/- 0.4 to 4.4 +/- 0.9. Bay K 8644 had no effect on the unitary conductance (27 pS in control), and relatively little effect in the open-close kinetic analysis. The mean open times were 4.2 ms and 5.2 ms, in control and Bay K 8644, respectively. These results suggest that the agonistic effect of Bay K 8644 involves a mechanism other than open-time prolongation, such as activation of silent channels.

Co-cultures of rat ventricular myocytes and sympathetic neurons were established. Superior cervical ganglia and ventricles from newborn rats were enzymatically dissociated and plated in a culture dish. Experiments were done between the 3rd (when evidence of neuron-myocyte proximity arises) and the 5th day in culture (before the myocytes become confluent). Simultaneous intracellular recording from a cardiomyocyte and an attached neuron was done using conventional microelectrode techniques (resistance of 60-100 Mohm). The myocytes in co-culture were either quiescent or spontaneously contracting. The contracting cells were either latent pacemaker or ventricular-like myocytes. The action potential (AP) characteristics of cardiomyocytes in co-cultures were comparable to those recorded in cardiomyocytes in pure cultures. Sympathetic innervation of the cardiomyocytes in co-cultures was evidenced by stimulating the neuron and observing an increase in rate of beating in latent pacemaker myocytes (average increase of 19.4 +/- 4.6%). In quiescent cardiomyocytes, neural stimulation evoked a slow depolarization that can reach threshold and initiate APs in the cell. This response is similar to slow excitatory postsynaptic potentials (EPSPs) observed in other synapses. Slow ESPSs could also be recorded in spontaneous beating cells, made quiescent by nifedipine (1x10(-6)-1x10(-7) M). These results indicate that functional synaptic contacts are developed in co-culture of sympathetic neurons and cardiac myocytes, and slow EPSPs can be evoked in cardiomyocytes as well as in other excitable cells. The sympathetic innervation occurring in culture did not significantly modify the spontaneous AP characteristics of the cardiomyocytes.

Studies in isolated preparations dealing with myocardial effects of catecholamines usually employ epinephrine concentrations 10-1,000 times higher (10(-7)-10(-5) M) than those observed during maximal cardiac adrenoceptor activation in vivo (10(-9)-5 x 10(-8) M) to obtain measurable cardiac responses. The reason for this discrepancy is still unclear, but it may reflect a diminished sensitivity to catecholamines in vitro. The main purpose of this study was to evaluate if a different myocardial sensitivity to epinephrine in vivo and in vitro does exist and to investigate which epinephrine concentrations in vitro mimic the effect of cardiac adrenoceptor activation in vivo. We compared concentration-response curves to cumulative increasing concentrations of of epinephrine, measured by high pressure liquid chromatography, in chloralose anesthetized or pithed rats (in vivo) and in isolated Langendorff perfused rat hearts (in vitro). We found that the amplitude of response to epinephrine was significantly higher in vivo at all concentrations. For example, an increase of 50 beats/min was observed at an epinephrine concentration of 29 +/- 6 nM in chloralose anesthetized, 25 +/- 4 nM in pithed rats and 149 +/- 52 nM in isolated hearts (P less than 0.05 vs. in vivo). Data on contractility closely parallel those on heart rate. These data indicate that, when methodological differences are minimized, there is a marked reduction in the amplitude of the response to epinephrine in vitro.(ABSTRACT TRUNCATED AT 250 WORDS)

The acute cardiac toxicity of three second-generation anthracycline analogues and doxorubicin was compared in a model of the rat isolated Langendorff perfused heart. The drugs, doxorubicin (DX), 4-epi-doxorubicin (4'EDX), 4-demethoxy-daunorubicin (4DMDR) and 4'-deoxy-doxorubicin (4'dxDX) were infused for 40 min at a concentration of 26 microM into the isolated hearts. All four compounds significantly reduced cardiac work and its first derivative. The time to 50% decrease in work (TW50) was respectively 36, 23, 9 and 7 min for DX, 4'EDX, 4'dxDX and 4DMDR. The three anthracycline derivatives, but not DX, significantly increased coronary resistance. Heart rate was reduced by all compounds compared with baseline, but not compared with controls. Rhythm disturbances were seen with all five hearts perfused with 4DMDR, which stopped beating before 40 min; 2/5 hearts in the 4'EDX group and 1/5 hearts in the 4'dxDX group also stopped before the end of perfusion. All the compounds reached concentrations in the myocardium 8 to 50 times higher than in the perfusing medium. The more cardiotoxic the compound, the higher was its myocardial concentration; a significant correlation was found for all four agents. Noradrenaline was never measurable in the perfusate of control and DX hearts; perfusion with the three anthracycline derivatives caused some release, but the pattern was not clearcut and the maximum concentrations attained in the perfusate were relatively low (less than or equal to 1.6 X 10(-9) M). In conclusion, in the rat isolated perfused heart, the early cardiotoxicity induced by equimolar concentrations of the three anthracycline compounds was greater than that induced by DX and was directly related to drug accumulation in the myocardium. Catecholamines do not seem to have a major role in the development of toxicity in this model.