Alterations in pulmonary vascular function in rats exposed to intermittent hypoxia

Vasoactive agents were examined in arteries from control rats and rats exposed to intermittent hypoxia (10% oxygen; 8 h/day) for 3, 5 or 20 days. Hypoxic rats developed right ventricular hypertrophy after 5 days, but became pulmonary hypertensive (elevated right ventricular systolic pressure; RVSP) only after 20 days. In pulmonary arteries (main and intralobar), responses to acetylcholine and ionomycin (endothelium-dependent vasodilators) were reduced after 20 and 5 days of intermittent hypoxia, whereas contractions to 5-hydroxytryptamine (5-HT) were enhanced (potency increase >10-fold) after 20, 5 and 3 days. Contractions to endothelin-1 and a thromboxane-mimetic, but not Ca-2divided by, were also increased. No changes in vascular function occurred in aorta. Since changes in pulmonary vascular function preceded the increase in RVSP they do not result from, but may contribute to, the development of hypoxia-induced pulmonary hypertension. If similar changes occur in humans, they may be important in conditions characterised by intermittent, as opposed to continuous, hypoxia. (C) 2003 Elsevier B.V. All rights reserved.

Respiratory and cardiovascular responses to hypoxia in the Australian lungfish

Simultaneous measurements of pulmonary blood flow (qPA), coeliacomesenteric blood flow (qCoA), dorsal aortic blood pressure (PDA), heart rate (fH) and branchial ventilation frequency (fv) were made in the Australian lungfish, /Neoceratodus forsteri, /during air breathing and aquatic hypoxia. The cho­linergic and adrenergic influences on the cardiovascular system were investigated during normoxia using pharmacological agents, and the presence of catecholamines and serotonin in different tissues was investi­gated using histochemistry. Air breathing rarely occurred during normoxia but when it did, it was always associated with increased pulmonary blood flow. The pulmonary vasculature is influenced by both a cho­linergic and adrenergic tonus whereas the coeliacomesenteric vasculature is influenced by a β-adrenergic vasodilator mechanism. No adrenergic nerve fibers could be demonstrated in /Neoceratodus /but catecholamine-containing endothelial cells were found in the atrium of the heart. In addition, serotonin-­immunoreactive cells were demonstrated in the pulmonary epithelium. The most prominent response to aquatic hypoxia was an increase in gill breathing frequency followed by an increased number of air breaths together with increased pulmonary blood flow. It is clear from the present investigation that /Neoceratodus /is able to match cardiovascular performance to meet the changes in respiration during hypoxia.

Identification of two novel mutations in the hydroxymethylbilane synthase gene in three patients from two unrelated families with acute intermittent porphyria

We have screened the hydroxymethylbilane synthase cDNAs of 3 patients from 2 families suffering from acute intermittent porphyria (AIP) from Scotland and South Africa using heteroduplex and chemical cleavage of mismatch analyses, Direct sequencing was used to characterise the mutations, The two novel mutations identified were a missense mutation at nucleotide position 64 in exon 3 (R22C) and a single base-pair deletion in exon 15, These mutations are predicted to affect the normal function of the enzyme and, therefore, are expected to be the primary cause of disease in these patients.

Muscle metabolites and performance during high-intensity, intermittent exercise

Six men were studied during four 30-s all-out exercise bouts on an air-braked cycle ergometer. The first three exercise bouts were separated by 4 min of passive recovery; after the third bout, subjects rested for 4 min, exercised for 30 min at 30-35% peak O-2 consumption, and rested for a further 60 min before completing the fourth exercise bout. Peak power and total work were reduced (P < 0.05) during bout 3 [765 +/- 60 (SE) W; 15.8 +/- 1.0 kJ] compared with bout 1 (1,168 +/- 55 mT, 23.8 +/- 1.2 kJ), but no difference in exercise performance was observed between bouts 1 and 4 (1,094 +/- 64 W, 23.2 +/- 1.4 kJ). Before bout 3, muscle ATP, creatine phosphate (CP), glycogen, pH, and sarcoplasmic reticulum (SR) Ca2+ uptake were reduced, while muscle lactate and inosine 5'-monophosphate were increased. Muscle ATP and glycogen before bout 4 remained lower than values before bout I (P < 0.05), but there were no differences in muscle inosine 5'-monophosphate, lactate, pH, and SR Ca2+ uptake. Muscle CP levels before bout 4 had increased above resting levels. Consistent with the decline in muscle ATP were increases in hypoxanthine and inosine before bouts 3 and 4. The decline in exercise performance does not appear to be related to a reduction in muscle glycogen. Instead, it may be caused by reduced CP availability, increased H+ concentration, impairment in SR function, or some other fatigue-inducing agent.

Acute intermittent porphyria: alternative splicing of hydroxymethylbilane synthase mRNA excludes exons 3 and 12

The hydroxymethylbilane synthase (HMBS) mRNAs from 44 control individuals and 30 patients suffering from acute intermittent porphyria (AIP), were screened for length differences by reverse transcriptase polymerase chain reaction (RT-PCR) and any abnormalities were characterized by direct sequencing. Examination of the mRNAs extracted from the peripheral blood lymphocytes of the samples revealed varying degrees of alternative splicing, involving the removal of exons 3 and 12. Approximately 10-50% of the mRNA molecules were affected, despite the absence of genomic splice site mutations or any major deviance from consensus splice sequence values. The preliminary data obtained from this study suggest that this event is a normal occurrence in peripheral blood lymphocytes, and may not be associated with the molecular pathology responsible for AIP. (C) 1998 Academic Press Limited.

Hypoxia stimulates cerebral blood flow in the estuarine crocodile (Crocodylus porosus)

The effect of N-2 respiration on cerebral blood flow (CBF) velocity on the dorsal surface of cerebellum was examined in the estuarine crocodile, Crocodylus porosus, using epi-illumination microscopy. Twelve minutes of N-2 respiration resulted in a 126% increase in CBF velocity. N-2 respiration had no effect on blood pressure, indicating an underlying cerebral vasodilation. In addition, heart rate increased significantly. Systemic injections of aminophylline and the NO synthase (NOS) inhibitor nitro-L-arginine (L-NA) did not affect the hypoxia induced increase in CBF. We conclude that C. porosus responds to hypoxia with adenosine and nitric oxide (NO) independent cerebral vasodilation, and that this is likely to be a mechanism protecting the brain from energy deficiency during prolonged dives. (C) 1999 Elsevier Science Ireland Ltd. All rights reserved.

Intermittent heparinised saline flushes for maintaining indwelling peripheral and central intravenous catheters in diabetic dogs

Intermittent low-dose heparinised saline flushes were found to be efficacious for maintaining patency of indwelling peripheral and central intravenous catheters in diabetic dogs. The catheters were flushed with 1 mL of 1 U/mL heparinised saline every two hours immediately following blood sample collection, or every 12 hours when not being used for sampling. Central catheters were flushed with saline solution first to clear the line before instillation of the heparinised saline. Patency of 54/57 (95%) of the peripheral catheters and 30/32 (94%) of the central catheters was achieved for up to 36 hours and five days, respectively. No phlebitis, or local or systemic infections were observed and, in each case, catheter failure was attributable to obstruction or extravasation. It is unlikely that there will be any contraindications to this flushing technique and its introduction may improve intravenous catheter survival and reduce catheter-associated complications in hospitalised dogs.

Can metal complexes serve as hypoxia activated prodrugs? Investigations of a Co(III) complex of the MMP inhibitor marimastat

For many years proof that the hypoxic nature of malignant tumours can be used to selectively target anticancer drugs has been sought. Several classes of potential redox activated anticancer drugs have been developed to take advantage of the reducing environment resulting from the hypoxia. Drug complexes with redox active metal centres as carriers have been investigated, but have largely been employed with cytotoxic drugs that require release of the drug intracellularly, complicating the design of such complexes. MMP inhibitors, a new class of anticancer drug, conversely act in the extracellular environment and we have investigated inhibitor complexes with several redox active transition metals. Marimastat is an MMP inhibitor with potent in-vitro antimetastatic activity and was recently in Phase III clinical trials for a variety of cancer types. We have synthesised a Co(II1) complex of marimastat incorporating the tetradentate ligand tpa (tris(2-methylpyridyl)amine) as a carrier ligand. The complex was structurally characterised in the solid state by single crystal X-ray diffraction, the first example of a crystal structure containing marimastat. 2D COSY and NOESY NMR spectra showed that the complex exists in two isomeric forms in solution, corresponding to the cis and trans isomers yet only crystallises in one of these forms. Biological testing of the complex in mice with 4T1.2 tumours showed interesting and unexpected outcomes. Initial results of the tumour growth inhibition study showed that a significant inhibition of growth was exhibited by the complex over the free inhibitor and the control. However, the metastatic potential of both free marimastat and the complex were higher than the control indicating likely problems with the experimental protocol. Further experiments are needed to determine the potential of such complexes as hypoxia activated prodrugs but there appears at least to be some promise.

Acute intermittent porphyria: the in vitro expression of mutant hydroxymethylbilane synthase

Acute intermittent porphyria (AIP) is an inborn error of haem biosynthesis caused by a variety of mutations in the gene coding for hydroxymethylbilane synthase (HMB-S). The entire coding sequence of this gene, from each of three South African AIP patients, was therefore screened for mutations using chemical cleavage mismatch (CCM) analysis and any changes detected characterized by DNA sequencing. Three single base changes were identified; a G(77) to A in exon 3, a C-346 to T in exon 8 and a G(518) to A in exon 10. These missense mutations, previously reported to be present in other populations, are known to be responsible for the structurally deleterious amino acid replacements R26H, R116W and R173Q, respectively. The in vitro expression of the enzymes containing these mutations and the subsequent measurement of their specific activities revealed a reduction to approximately 4% of normal activity. (C) 1997 Academic Press Limited.

An improved survival model of hypoxia/ischaemia in the piglet suitable for neuroprotection studies

The purpose of this study, was to develop a newborn piglet model of hypoxia/ischaemia which would better emulate the clinical situation in the asphyxiated human neonate and produce a consistent degree of histopathological injury following the insult. One-day-old piglets (n = 18) were anaesthetised with a mixture of propofol (10 mg/kg/h) and alfentinal (5,5.5 mug/kg/h) i.v. The piglets were intubated and ventilated. Physiological variables were monitored continuously. Hypoxia was induced by decreasing the inspired oxygen (FiO(2)) to 3-4% and adjusting FiO(2) to maintain the cerebral function monitor peak amplitude at less than or equal to5 muV. The duration of the mild insult was 20, min while the severe insult was 30 min which included 10 min where the blood pressure was allowed to fall below 70% of baseline. Control piglets (n=4 of 18) were subjected to the same protocol except for the hypoxic/ischaemic insult. The piglets were allowed to recover from anaesthesia then euthanased 72 It after the insult. The brains were perfusion-fixed, removed and embedded in paraffin. Coronal sections were stained by haematoxylin/eosin. A blinded observer examined the frontal and parietal cortex, hippocampus, basal ganglia, thalamus and cerebellum for the degree of damage. The total mean histology score for the five areas of the brain for the severe insult was 15.6 +/-4.4 (mean +/-S.D., n=7), whereas no damage was seen in either the mild insult (n=4) or control groups. This 'severe damage' model produces a consistent level of damage and will prove useful for examining potential neuroprotective therapies in the neonatal brain. (C) 2001 Elsevier Science BY. All rights reserved.

Chronic hypoxia induced down-regulation of angiotensinogen expression in rat epididymis

The presence of an intrinsic renin-angiotensin system (RAS) in the rat epididymis has been previously established by showing the expression of several key RAS components, and in particular angiotensinogen, the indispensable element for the intracellular generation of angiotensin II. In this study, the possible involvement of this local epididymal RAS in the testicular effects of chronic hypoxia was investigated. Semi-quantitative reverse-transcription polymerase chain reaction (RT-PCR), Western blotting and by in situ hybridization histochemistry of the rat epididymis were used to show changes in localization and expression of angiotensinogen. Results from RT-PCR analysis demonstrated that chronic hypoxia caused a marked decrease (60%) in the expression of angiotensinogen mRNA, when compared with that in the normoxic epididymis. Western blot analysis demonstrated a less decrease (35%) in the expression of angiotensinogen protein. In situ hybridization histochemistry showed that the reduced angiotensinogen mRNA in chronic hypoxia was specifically localized to the epididymal epithelium from the cauda, corpus and caput regions of the epididymis; a distribution similar to that of normoxic rats. It was concluded that chronic hypoxia decreases the transcriptional and translational expression of angiotensinogen, and thus local formation of angiotensin II, in the rat epididymis. (C) 2001 Elsevier Science B.V. All rights reserved.

Pharmacotherapy of intermittent claudication

Intermittent claudication (IC) is leg muscle pain, cramping and fatigue brought on by exercise and is the primary symptom of peripheral arterial disease. The goals of pharmacotherapy for IC are to increase the walking capacity/quality of life and to decrease rates of amputation. In 1988, pentoxifylline was the only drug that had reasonable supportive clinical trial evidence for being beneficial in IC. Since then a number of drugs have shown benefit or potential in IC. Cilostazol, a specific inhibitor of phosphodiesterase 3 and activator of lipoprotein lipase, clearly increases pain-free and absolute walking distances in claudicants. However, cilostazol does cause minor side effects including headache, diarrhoea, loose stools and flatulence. Naftidrofuryl, a serotonin (5-HT2) receptor antagonist and antiplatelet drug, is beneficial in claudicants. Inhibitors of platelet aggregation (including nitric oxide from L-arginine or glyceryl trinitrate) and anticoagulants (low molecular weight heparin, defibrotide) probably have both short and long-term benefits in IC. In addition, intravenous infusions of prostaglandins (PGs) PGE1 and PGI2 have an established role in severe peripheral arterial disease and the recent introduction of longer lasting and/or oral forms of the PGs makes them more likely to be useful in the IC associated with less severe forms of the disease. There are some exciting new approaches to the treatment of IC, including propionyl-L-carnitine and basic fibroblast growth factor (bFGF).

Physiological and symptomatic responses to cycling and walking in intermittent claudication

To shed light on the potential efficacy of cycling as a resting modality in the treatment of intermittent claudication (IC), this study compared physiological and symptomatic responses to graded walking and cycling tests in claudicants. Sixteen subjects with peripheral arterial disease (resting ankle:brachial index (ABI) < 0.9) and IC completed a maximal graded treadmill walking (T) and cycle (C) Lest after three familiarization tests on each mode. During cacti test, symptoms, oxygen uptake (VO2), minute ventilation (V-E), (respiratory exchange ratio) (RER) and heart rate (HR) were measured, and for 10 min after each Lest the brachial and ankle systolic pressures were recorded, All but One subject experienced calf pain as the primary limiting symptom during T whereas the symptoms were more varied during C and included thigh pain, calf pain and dyspnoea, Although maximal exercise time was significantly longer on C than T (690 +/- 67 vs, 495 +/- 57 s), peak VO2, peak, V-E and peak heart rate during C and T were not different; whereas peak RER was higher during C. These responses during C and T were also positively 1, (P < 0.05) with each other, with the exception of RER. The postexercise systolic pressures were also not different between C and T. However, the peak decline ill ankle pressures from resting values after C and T were not correlated with each other. Thew data demonstrate that cycling and walking induce a similar level of metabolic and cardiovascular strain, but that the primary limiting symptoms and haemodynamic response in an individual's extremity, measured after exercise, can differ substantially between these two modes.