Development and further characterization of a small subclass of rat olfactory receptor neurons that shows immunoreactivity for the HSP70 heat shock protein

We previously described a rat olfactory receptor neuron (ORN) subpopulation [the 2A4(+) ORNs] that shows uniquely strong reactivity with antibodies to the 70-kD heat shock protein (HSP70) family of molecular chaperones (Carr et al. [1994] J. Comp. Neurol. 348:150-160). The 2A4(+)ORNs are dispersed through zones II-IV of the olfactory epithelium (OE), and their axons project to only two or three glomeruli that are located consistently in each olfactory bulb (OB). To date, the 2A4(+)ORN subpopulation is the only cell population to show such distinct HSP70 immunoreactivity as well as the most discrete ORN subpopulation to be so labeled. The present report shows that 2A4(+)ORN neurons first appear between postnatal days 7 (P7) and P10. Initially, low cell numbers rise to a density of 0.1 2A4(+)ORNs/mm OE length by P14, plateau at 0.9 2A4(+)ORNs/mm by P49, then fall to adult values of 0.4 cells/mm. Autoradiographic birthdating indicates that almost all of these early appearing 2A4(+)ORNs are generated postnatally, in contrast to the prenatal generation of all ORN subpopulations characterized to date by their expression of olfactory receptor protein mRNAs. A developmentally related increase in the mean depth of 2A4(+)ORNs within the OE also occurs. In the OB, initial 2A4(+)axonal projections are to only two or three glomeruli, as in adults. Slight but significant rostral shifts in (+)glomerular location occur with development. The 2A4(+)ORN immunoreactivity was found to be due to expression of HSP70, the dominant stress-inducible member of the HSP70 family, rather than constitutively expressed HSC70. In addition, despite their presence in rat OE, no 2A4(+)ORNs were found in mice, gerbils, guinea pigs, or hamsters.     

Phosphorylation of cytosolic phospholipase A2 and the release of arachidonic acid in human neutrophils

Kinases mediating phosphorylation and activation of cytosolic phospholipase A2 (cPLA2) in intact cells remain to be fully characterized. Platelet-activating factor stimulation of human neutrophils increases cPLA2 phosphorylation. This increase is inhibited by PD 98059, a mitogen-activated protein (MAP)/extracellular signal-regulating kinase (erk) 1 inhibitor, but not by SB 203580, a p38 MAP kinase inhibitor, indicating that this action is mediated through activation of the p42 MAP kinase (erk2). However, platelet-activating factor-induced arachidonic acid release is inhibited by both PD 98059 and SB 203580. Stimulation by TNF-alpha increases cPLA2 phosphorylation, which is inhibited by SB 203580, but not PD 98059, suggesting a role for p38 MAP kinase. LPS increases cPLA2 phosphorylation and arachidonic acid release. However, neither of these actions is inhibited by either PD 98059 or SB 203580. PMA increases cPLA2 phosphorylation. This action is inhibited by PD 98059 but not SB 203580. Finally, FMLP increases cPLA2 phosphorylation and arachidonic acid release. Interestingly, while the FMLP-induced phosphorylation of cPLA2 is not affected by the inhibitors of the p38 MAP kinase or erk cascades, both inhibitors significantly decrease arachidonic acid release stimulated by FMLP. SB 203580 or PD 98059 has no inhibitory effects on the activity of coenzyme A-independent transacylase.     

Plasma marinobufagenin-like and ouabain-like immunoreactivity during saline volume expansion in anesthetized dogs

OBJECTIVES: This study investigated effects of acute plasma volume expansion on plasma levels and urinary output of two endogenous Na,K-ATPase inhibitors, marinobufagenin-like and ouabain-like immunoreactive substances. METHODS: Plasma volume was expanded for 3 h via intravenous saline infusion in three groups of anesthetized dogs--nontreated (n = 5); pretreated with rabbit antidigoxin (n = 5); and pretreated with rabbit antimouse (control) antibody (n = 4). RESULTS: Plasma marinobufagenin-like immunoreactivity increased to 11.87 +/- 3.16 nmol.l-1 (vs. 0.30 +/- 0.16 nmol.l-1) within 10 min of volume expansion, in parallel with a 15% increase in LVdP/dt, then decreased to 2.21 +/- 0.59 nmol.l-1, and in 90 min increased to 11.8 +/- 2.8 nmol.l-1, in parallel with the maximal natriuretic response. Plasma concentrations of ouabain-like immunoreactive material were increased after 90 min of saline infusion (0.019 +/- 0.004 nmol.l-1 vs. 0.139 +/- 0.056 nmol.l-1). Pretreatment of the animals with antidigoxin antibody blocked the positive inotropic and reduced natriuretic response to volume expansion, and decreased the urinary release of marinobufagenin-like, but not ouabain-like, material. CONCLUSIONS: These results show the presence of marinobufagenin-like immunoreactive substance in dog plasma and suggest that mammalian EDLF may have a bufodienolide nature. Endogenous marinobufagenin-like immunoreactive substance, which is likely to cross-react with antidigoxin antibody, is involved in the natriuretic and positive inotropic responses to plasma volume expansion.     

Acute hypoxic pulmonary vasoconstriction in man is attenuated by type I angiotensin II receptor blockade

OBJECTIVES: We examined the hypothesis that angiotensin II (ANG II) is a modulator of acute hypoxic pulmonary vasoconstriction (HPV) by looking at the effect of losartan, a selective type 1 ANG II receptor antagonist, on acute HPV in man. METHODS: Ten normal volunteers were studied on two separate days. They either received pre-treatment with losartan 25, 50, 100, 100 mg respectively on four consecutive days or matched placebo. They were then rendered hypoxaemic, by breathing an N2/O2 mixture for 20 min to achieve an SaO2 of 85-90% adjusted for a further 20 min to achieve an SaO2 of 75-80%. Pulsed wave Doppler echocardiography was used to measure mean pulmonary artery pressure (MPAP), cardiac output and hence pulmonary vascular resistance (PVR). RESULTS: Baseline MPAP and PVR (during normoxaemia) were unaffected by losartan pre-treatment compared with placebo. However, losartan significantly reduced MPAP at both levels of hypoxaemia compared with placebo: 14.7 +/- 0.7 vs 19.0 +/- 0.7 mmHg at an SaO2 85-90% (P < 0.01) and 20.0 +/- 0.7 vs 25.7 +/- 0.8 mmHg at an SaO2 75-80% (P < 0.05) respectively. Similarly losartan significantly reduced PVR compared to placebo: 191 +/- 9 vs 246 +/- 10 dyne.s.cm-5 at an SaO2 85-90% (P < 0.005) and 233 +/- 12 vs 293 +/- 18 dyne.s.cm-5 at an SaO2 75-80% (P < 0.05), respectively. Pre-treatment with losartan, however, had no significant effect on systemic vascular resistance although losartan compared to placebo resulted in a significant (P < 0.05) reduction in mean arterial pressure at an SaO2 75-80%: 78 +/- 2 vs 87 +/- 2 mmHg. CONCLUSIONS: Losartan had no effect on baseline pulmonary haemodynamics but significantly attenuated acute hypoxic pulmonary vasoconstriction, suggesting that angiotensin II plays a role in modulating this response in man via its effects on the type 1 angiotensin II receptor.     

Liposome-mediated therapy of intracranial brain tumors in a rat model

PURPOSE: Malignant brain tumors represent a serious therapeutic challenge, and survival often is low. We investigated the delivery of doxorubicin (DXR) to rat brain tumors in situ via liposomes, to test the hypothesis that intact liposomes undergo deposition in intracranial tumor through a compromised blood-tumor vasculature. Both therapeutic effect and intra-tumor drug carrier distribution were evaluated to identify variables in carrier-mediated delivery having impact on therapy. METHODS: The rat 9L gliosarcoma tumor was implanted orthotopically in Fischer 344 rats in the caudate-putamen region. The tumor-bearing rats were treated with DXR, either free or encapsulated in long-circulating, sterically-stabilized liposomes. Anti-tumor efficacy was assessed by survival time. In parallel, liposomes labeled with a fluorescent phospholipid analog were injected into tumor-bearing rats. At predetermined intervals, the brains were perfused with fixative, sectioned, and imaged with laser scanning confocal microscope (LSCM) to investigate the integrity of the tumor vascular bed and the intratumor deposition of liposomes. RESULTS: Free DXR given in 3 weekly iv injections was ineffective in increasing the life span of tumor-bearing rats at cumulative doses < or = 17 mg/kg, and at the highest dose (17 mg/kg) decreased survival slightly, compared to saline-treated controls. In contrast, DXR encapsulated in long-circulating liposomes mediated significant increases in life span at 17 mg/kg. Rats showed a 29% percent increase in median survival, respectively, compared to saline-control animals. The delay of treatment after tumor implantation was a major determinant of therapeutic effect. Fluorescent liposomes were deposited preferentially in tumor rather than normal brain, and were distributed non-uniformly, in close proximity to tumor blood vessels. CONCLUSIONS: Liposomes can be used to enhance delivery of drugs to brain tumors and increase therapeutic effect. The therapeutic effect may arise from release of drug from liposomes extravasated in discrete regions of the tumor vasculature and the extravascular space.     

Differential scanning microcalorimetry indicates that human defensin, HNP-2, interacts specifically with biomembrane mimetic systems

alpha-Defensins are antimicrobial peptides with 29-35 amino acid residues and cysteine-stabilized amphiphilic, triple-stranded beta-sheet structures. We used high-precision differential scanning microcalorimetry to investigate the effects of a human neutrophil alpha-defensin, HNP-2, on the phase behavior of model membranes mimicking bacterial and erythrocyte cell membranes. In the presence of this positively charged peptide, the phase behavior of liposomes containing negatively charged phosphatidylglycerol was markedly altered even at a high lipid-to-peptide molar ratio of 500:1. Addition of HNP-2 to liposomes mimicking bacterial membranes (mixtures of dipalmitoylphosphatidylglycerol and -ethanolamine) resulted in phase separation owing to some domains being peptide-poor and others peptide-rich. The latter are characterized by an increase of the main transition temperature, most likely arising from electric shielding of the phospholipid headgroups by the peptide. On the other hand, HNP-2 did not affect the phase behavior of membranes mimicking erythrocyte membranes (equimolar mixtures of dipalmitoylphosphatidylcholine and sphingomyelin) as well as the pure single components. This is in contrast to melittin, which significantly affected the phase behavior of choline phospholipids in accordance with its unspecific lytic activity. These results support the hypothesis of preferential interaction of defensins with negatively charged membrane cell surfaces, a common feature of bacterial cell membranes, and demonstrate that HNP-2 discriminates between model membrane systems mimicking prokaryotic and eukaryotic cell membranes.