Determination of cell adhesion sites of neuropilin-1.

Neuropilin-1 is a type 1 membrane protein with three distinct functions. First, it can mediate cell adhesion via a heterophilic molecular interaction. Second, in neuronal cells, neuropilin-1 binds the class 3 semaphorins, which are neuronal chemorepellents, and plays a role in the directional guidance of axons. Neuropilin-1 is expected to form complexes with the plexinA subfamily members and mediate the semaphorin-elicited inhibitory signals into neurons. Third, in endothelial cells, neuropilin-1 binds a potent endothelial cell mitogen, vascular endothelial growth factor (VEGF)(165), and regulates vessel formation. Though the binding sites in neuropilin-1 for the class 3 semaphorins and VEGF(165) have been analyzed, the sites involved in cell adhesion activity of the molecule have not been identified. In this study, we produced a variety of mutant neuropilin-1s and tested their cell adhesion activity. We showed that the b1 and b2 domains within the extracellular segment of neuropilin-1 were required for the cell adhesion activity, and peptides with an 18-amino acid stretch in the b1 and b2 domains were sufficient to induce the cell adhesion activity. In addition, we demonstrated that the cell adhesion ligands for neuropilin-1 were proteins and distributed in embryonic mesenchymal cells but distinct from the class 3 semaphorins, VEGF, or plexins.

Increased sensitivity of neonate atrial myocytes to adenosine A1 receptor stimulation in regulation of the L-type Ca2+ current.

Adenosine has cardioprotective effects against ischemia, and newborn hearts show high resistance to ischemia. The effects of purinoceptor stimulation by adenosine and ATP on the L-type Ca2+ current (ICa) were examined in atrial cells from neonate and adult rabbits. ICa was measured by the membrane-perforated patch method. Adenosine inhibited the isoproterenol-stimulated ICa more potently in neonate cells than in adult cells. The high sensitivity of neonate myocytes to adenosine was accompanied not only by an increased maximum response but also by a lower IC50 concentration. ATP also inhibited isoproterenol-stimulated ICa. The effect of ATP on neonate cells was stronger than that on adult cells at high concentrations (greater than or = 100 microM). The effect of adenosine was antagonized by an A1 adenosine receptor antagonist, 1,3-dipropyl-8-cyclopentylxanthine (DPCPX). DPCPX or an ecto-5'-nucleosidase inhibitor (alpha,beta-methylene-ADP) blocked most (approximately 60%) of the effect of ATP (30 microM), and co-addition of DPCPX and suramin (P2 receptor blocker) abolished the effect of ATP. Suramin alone did not reduce the effect of ATP significantly in neonate cells. Both the effects of adenosine and ATP were eliminated by pre-treatment with pertussis toxin or by superfusion with forskolin plus 3-isobutyl-1-methylxanthine (IBMX). Inhibitors of the nitric oxide-cyclic GMP pathway did not affect the adenosine inhibition of ICa. In summary, neonatal myocardial cells are highly sensitive to adenosine A1 receptor stimulation. ATP stimulates both the adenosine A1 and P2 receptors. Adenosine A1 receptor stimulation, as a result of hydrolysis of ATP, predominantly mediates the effect of ATP, and the role of P2 receptors in the ATP inhibition of ICa is relatively small in neonate cells. The high sensitivity to adenosine may contribute to the ischemic tolerance of newborn hearts.

Modulation by chloramine-T of 4-aminopyridine-sensitive transient outward current in rabbit atrial cells.

The effects of an oxidizing agent, chloramine-T, on the 4-aminopyridine-sensitive transient outward current (ITO) were investigated in rabbit atrial myocytes by using patch-clamp techniques. Extracellular application of chloramine-T at 20 microM irreversibly slowed the time course of inactivation of the whole-cell ITO, and increased the peak by 19.3% (n = 19) at +40 mV. At 100 microM, chloramine-T decreased the peak by 22.5% (n = 9) of the control, and subsequently induced a glibenclamide-sensitive time-independent outward K+ current. Under superfusion with dithiothreitol (3 mM), chloramine-T (100 microM) produced no change in ITO. The chloramine-T-induced slowing of ITO inactivation was partially reversed by subsequent application of 3 mM dithiothreitol. In single-channel recordings with the cell-attached patch configuration, chloramine-T (20 microM) increased the open probability of the ITO channel from 0.15 to 0.46 at a potential 100 mV positive to the resting potential, and the mean open lifetime from 5.1 ms to 7.0 ms (n = 5). The unitary current amplitude was not affected. As a result, chloramine-T increased the ensemble current in amplitude and slowed its decay. These results indicated that: (1) inactivation of the native A-type channels of rabbit heart is susceptible to oxidation; and (2) oxidation of ITO channels may contribute to the genesis of arrhythmias.

Effects of hydrogen peroxide on the transient outward current in rabbit atrial myocytes.

1. In the present study, we investigated the effects of hydrogen peroxide (H2O2) on the 4-aminopyridine-sensitive transient outward current (I(TO)) in rabbit atrial myocytes using the amphotericin B-perforated patch voltage-clamp method. 2. Superfusion of myocytes with H2O2 at 100 micromol/L gradually slowed the time-course of inactivation of I(TO) and increased the peak by 9% (n = 9). The H2O2-induced slowing of I(TO) inactivation was concentration dependent (over the concentration range 10 micromol/L to 1 mmol/L). These effects were hardly reversed by washout of H2O2, but were quickly abolished by dithiothreitol (2 mmol/L). 3. Bisindolylmaleimide (100 nmol/L), an inhibitor of protein kinase C, significantly attenuated the H2O2-induced effects on I(TO). 4. These results suggest that rabbit atrial I(TO) is susceptible to oxidation by H2O2 at concentrations relevant to those encountered during ischaemia/reperfusion and that protein kinase C modulates the effects of H2O2.

Chromosome assignment of four plexin A genes (Plxna1, Plxna2, Plxna3, Plxna4) in mouse, rat, Syrian hamster and Chinese hamster.

We determined chromosome locations of four plexin A subfamily genes, Plxna1, Plxna2, Plxna3 and Plxna4, in four rodent species, mouse, rat, Syrian hamster and Chinese hamster, by fluorescence in situ hybridization. Plxna1, Plxna2, Plxna3 and Plxna4 were localized to Chr 6E2, 1H6, XB-C1 and 6B1 in mouse, Chr 4q34.1, 13q26-->q27, Xq37.1-->q37.2 and 4q21.3-->q22 in rat, Chr 8qb1.1-->qb1.3, 11qb8, Xpb8 and 5qb3.3 in Syrian hamster, and Chr 8q1.2, 5q3.7, Xp2.7 and 1q2.2-->q2.3 in Chinese hamster, respectively. All the mouse and rat plexin A genes were localized to chromosome regions where conserved homology has been identified among human, mouse and rat.

Differential expression of plexin-A subfamily members in the mouse nervous system.

Plexins comprise a family of transmembrane proteins (the plexin family) which are expressed in nervous tissues. Some plexins have been shown to interact directly with secreted or transmembrane semaphorins, while plexins belonging to the A subfamily are suggested to make complexes with other membrane proteins, neuropilins, and propagate chemorepulsive signals of secreted semaphorins of class 3 into cells or neurons. Despite that much information has been gathered on the plexin-semaphorin interaction, the role of plexins in the nervous system is not well understood. To gain insight into the functions of plexins in the nervous system, we analyzed spatial and temporal expression patterns of three members of the plexin-A subfamily (plexin-A1, -A2, and -A3) in the developing mouse nervous system by in situ hybridization analysis in combination with immunohistochemistry. We show that the three plexins are differentially expressed in sensory receptors or neurons in a developmentally regulated manner, suggesting that a particular plexin or set of plexins is shared by neuronal elements and functions as the receptor for semaphorins to regulate neuronal development.

EP receptor-mediated inhibition by prostaglandin E(1) of cardiac L-type Ca(2+) current of rabbits.

Prostaglandin E(1) (PGE(1)) has cardioprotective effects on the ischemic-reperfused heart. To clarify the mechanisms underlying the protective action of PGE(1) on myocardium, we examined the effect of PGE(1) on the L-type Ca(2+) current (I(Ca)) using single atrial cells from rabbits. PGE(1) did not show a significant effect on basal I(Ca) but inhibited the I(Ca) prestimulated by isoproterenol (Iso, 30 nM). This inhibition was concentration dependent (EC(50) = 0.027 microM). Both sulprostone, a specific PGE receptor subtype (EP(1) and EP(3)) agonist, and 11-deoxy-PGE(1), an EP(3) agonist, inhibited the Iso-stimulated I(Ca), similar to PGE(1). Pretreatment with pertussis toxin (PTX) abolished the PGE(1) inhibition of I(Ca). Both the application of forskolin plus IBMX and intracellular dialysis with 8-bromoadenosine 3',5'-cyclic monophosphate eliminated the effect of PGE(1). PGE(1) did not show any further inhibition of I(Ca) when the effect of Iso was almost fully antagonized by acetylcholine. Methylene blue (guanylate cyclase inhibitor), KT-5823 (cGMP-dependent protein kinase inhibitor), and erythro-9-(2-hydroxy-3-nonyl)adenine (type II phosphodiesterase inhibitor) did not significantly change the inhibitory effect of PGE(1). These findings suggest that 1) PGE(1) inhibits Iso-stimulated I(Ca) by binding to the EP(3) receptor and 2) the PTX-sensitive and cAMP-dependent pathway is involved in the PGE(1) inhibition of I(Ca), but the nitric oxide-cGMP-dependent pathway is not. The PGE(1)-induced antiadrenergic effect shown in this study may contribute to the PGE(1) protection of myocardium against ischemia.

Endothelin-1 inhibits pacemaker currents in rabbit SA node cells.

Our recent study demonstrated that endothelin-1 (ET-1) inhibits the pacemaker activity of sinoatrial (SA) node cells via changes in the L-type Ca2+, delayed K+, and background K+ currents. Using the whole-cell patch-clamp technique in the same preparation, we found that ET-1 reduces other pacemaker currents, the T-type Ca2+ current (ICa,T) and the hyperpolarization-activated inward current (I(f)). The inhibitory actions of ET-1 on these currents were concentration-dependent, i.e., EC50 of 0.9 nM for ICa,T and 2.3 nM for I(f), with little reversal after washout of the peptide. In the presence of BQ485, both currents were not affected by ET-1. These results indicate additional mechanisms underlying negative chronotropic actions of ET-1 on the rabbit SA node.

Myocardial oxygen consumption, cardiac work, and myocardial efficiency in children.

UNLABELLED: Few reports on human cardiac functional development exist, although this information is important for managing paediatric heart disease. The work and the energy usage of the heart was measured in children. A total of 58 patients (aged 1-19 years) with a history of Kawasaki disease without coronary sequelae underwent cardiac catheterization to obtain haemodynamic data and to measure myocardial oxygen consumption. Myocardial oxygen consumption (ml/min) (y = 0.63 x + 3.6, r = 0.86, P < 0.0001, x = age) and left ventricular minute work (kg m/min) (y = 0.46 x 2.4, r = 0.84, P < 0.0001, x = age) correlated positively with age. However, left ventricular minute work per body surface area (age: 2-5 years, 5.8 +/- 0.34 kg m/min/m2; age: 6-10 years, 6.9 +/- 0.59 kg m/min/m2; age: 11-15 years, 5.9 +/- 0.51 kg m/min/m2; age 16-19 years, 6.5 +/- 0.29 kg m/min/m2; and myocardial efficiency (age: 2-5 years, 40.1 +/- 4.4%; age: 6-10 years, 42.4 +/-3.9%; age: 11-15 years, 45.9 +/- 4.1%; age: 15-19 years, 42.3 +/- 6.6%) remained constant throughout childhood. CONCLUSION: In spite of the structural immaturity of the developing heart, the myocardial oxygen consumption per body surface area and myocardial efficiency led by the cardiac work are the same in adults and in children older than 1 year of age.

Identification of plexin family molecules in mice.

By screening of E17.5 mouse brain cDNA libraries, we isolated two cDNAs encoding new plexin-like proteins. Sequencing revealed that these two proteins were type 1 membrane proteins which showed over 60% identity at the amino acid level to mouse plexin 1. Moreover, putative extracellular segments of these two proteins had three repeats of a cysteine-rich domain which is a common motif for plexin proteins. Thus, we named these two proteins mouse plexin 2 and mouse plexin 3. We obtained mouse plexin 3 cDNA clones in which a part of protein-coding region was deleted. Also, Northern blot analysis showed molecular heterogeneity in mouse plexin 2 mRNAs. These findings indicate that in the mouse, plexins comprise a molecular family (the plexin family).

Identification of a neuronal cell surface molecule, plexin, in mice.

We searched for mouse homologues of the cell adhesion protein plexin which was originally found in Xenopus, and obtained a cDNA encoding a plexin-like protein. We referred to this protein as mouse plexin 1. The overall amino acid identity between mouse plexin 1 and Xenopus plexin was 84%. As in the Xenopus plexin, the extracellular segment of mouse plexin 1 protein possessed three cysteine-rich domains which showed significant homology with the cysteine-rich domain of the c-Met proto-oncogene protein product (c-Met protein) and Met-like receptor protein tyrosine kinases. Northern blot analysis indicated that mouse plexin 1 was predominantly expressed in the brain.