bluuebunny/crossref_metadata_2025 huggingface dataset:
Imaging And Nano‐Manipulation Of Single Actomyosin Motors At Work
Authors: Ishii Yoshiharu , Kimura Yuji , Kitamura Kazuo , Tanaka Hiroto , Wazawa Tetsuichi , Yanagida Toshio
Published: March 2003

1. Muscle contraction is achieved by the sliding movement of myosin and actin using the energy of ATPase.

2. Our research has focused on the question of how chemical energy is used to perform mechanical work. Recent developments of single molecule imaging and manipulation techniques have allowed us to study the chemical and mechanical events at a molecular level.

3. There are many lines of evidence that show that the energy liberated from ATPase is stored in the actomyosin molecules for later use.

4. The displacement produced by a single ATP molecule is made up of several steps, each of 5.3 nm. Sometimes the mechanical event is delayed after the ADP is released from myosin.

5. This storage of energy may be explained by a slow conformational transition between the metastable states of the proteins. This suggestion has been supported by results obtained using single‐molecule fluorescence spectroscopy.
Effects Of β‐Adrenoceptor Stimulation On Pacing‐Induced Failure Of Dog Hypertrophic Hearts
Authors: Huang Mark W , Leone Richard J Leone , Weiss Harvey R , Tse James , Scholz Peter M
Published: March 2003

1. We tested the hypothesis that the transition to pacing‐induced failure in hypertrophic hearts would result in reduced functional and metabolic responses to β‐adrenoceptor stimulation.

2. Isoproterenol (ISO; 0.1 μg/kg per min) was infused into a coronary artery in five anaesthetized open‐chest control, five aortic stenosis‐induced left ventricular hypertrophy (LVH) and five LVH pacing‐induced failure dogs.

3. In both control and LVH dogs, but not in failure dogs, ISO significantly increased local regional work (1923±665 vs 2656±715, 1185±286 vs 1906±562 and 835±106 vs 849± 216 g.mm/min, respectively), force (11.1±1.4 vs 16.9±2.6, 8.6±1.5 vs 13.7±2.3 and 12.2±1.1 vs 11.0±1.8 g, respectively) and myocardial O2 consumption (7.3±2.0 vs 10.0±1.5, 8.2±1.6 vs 11.6±2.6 and 4.4±1.5 vs 5.5±1.8 mL O2/min per 100 g, respectively).

4. Isoproterenol also significantly increased cAMP in control and LVH dogs (474±67 vs 600±91 and 473±34 vs 619± 53 pmol/g, respectively). In heart failure, cAMP was significantly lower and there was no significant increase in cAMP in response to ISO (245±43 vs 314±40 pmol/g, respectively).

5. We conclude that there were no significant myocardial functional, O2 consumption or cAMP responses to ISO after the transition from hypertrophy to cardiac failure.
Pretreatment With Ramiprilat Induces Cardioprotection Against Free Radical Injury In Guinea‐Pig Isolated Heart: Involvement Of Bradykinin, Protein Kinase C And Prostaglandins
Authors: Jin Zhu‐Qiu , Chen Xiu
Published: March 2003

1. Pretreatment with ramiprilat, an angiotensin‐converting enzyme (ACE) inhibitor, induced cardioprotection and its possible mechanism of action was investigated in guinea‐pig Langendorff perfused heart.

2. Superoxide anion (•O2–), produced by hypoxanthine and xanthine oxidase, and the 1,1‐diphenyl‐2‐picryl‐hydrazyl (DPPH) free radical were used for triggering free radical injury in cardiac tissue.

3. 1,1‐Diphenyl‐2‐picryl‐hydrazyl and •O2– significantly reduced left ventricular developed pressure (LVDP), ±dP/dtmax, heart rate and coronary flow. Left ventricular end‐diastolic pressure (LVEDP) was elevated and lactate dehydrogenase (LDH) leakage and the formation of thiobarbituric acid‐reactive substances (TBARS) formation were significantly increased.

4. Pretreatment with ramiprilat induced cardioprotection against DPPH and •O2– free radical injury. Cardiac functions (LVDP, LVEDP and ±dP/dtmax) were significantly improved. Both LDH and TBARS were reduced.

5. HOE 140 (a selective bradykinin B2 receptor antagonist), calphostin C (a protein kinase C (PKC) inhibitor) and indomethacin (a cyclo‐oxygenase inhibitor) all abolished the cardiac protective effect of ramiprilat. However, NG‐nitro‐
L‐arginine methyl ester, a nitric oxide synthase inhibitor, had no effect.

6. In conclusion, ramiprilat pretreatment induces cardioprotection against either DPPH or •O2– free radical injury. The protective effect depends on activation of B2 receptors and PKC. Prostaglandin synthesis is also involved.
Tissue Distribution Of α‐Tocopherol In Nephrotic Rats
Authors: Medina‐Campos On , Mancera J , Pedraza‐Chaverrí J
Published: March 2003

1. Reactive oxygen species are involved in the pathogen‐ esis of puromycin aminonucleoside (PAN) nephrosis and α‐tocopherol is one of the major anti‐oxidants in the body.

2. In the present study, we measured the levels of α‐tocopherol by high‐performance liquid chromatography in the plasma and in nine tissues of control and nephrotic rats obtained 10 days after either 0.9% saline solution or PAN injection, respectively.

3. In nephrotic rats, α‐tocopherol levels increased four‐ fold in plasma; however, the molar ratio of α‐tocopherol/ cholesterol remained unchanged, suggesting that the increase in α‐tocopherol content was attributable to an increase in plasma lipid concentration.

4. In nephrotic rats, the α‐tocopherol/cholesterol ratio increased 1.33‐fold in adrenal glands and 1.34‐fold in the testis, but remained unchanged in heart, spleen, liver, kidney lung, brain and muscle.

5. These data suggest that, in PAN nephrotic rats, there are alterations in the distribution of α‐tocopherol and there is no deficiency of α‐tocopherol in plasma or tissues.
Presynaptic Inhibitory Actions Of Lignocaine In Canine Isolated, Blood‐Perfused Atrial Preparations
Authors: Tsuboi Masato , Chiba Shigetoshi
Published: March 2003

1. Cardiac effects of lignocaine on sinoatrial nodal pacemaker activity and atrial contractility were investigated in five canine isolated, blood‐perfused right atria that were perfused with heparinized blood from support dogs. The effects of lignocaine on responses to intracardiac nerve stimulation and administered acetylcholine and noradrenaline were also examined.

2. Lignocaine was injected into the support dog intravenously or administered selectively to the sinus node artery of the isolated atrium. At doses that did not produce significant depressor action (0.3, 1.0 and 3.0 mg/kg), lignocaine produced no significant changes in heart rate. A large dose of 10 mg/kg lignocaine caused significant depressor effects and slight bradycardia. Direct administration of lignocaine (0.3, 1.0, 3.0, 10.0 and 30.0 μmol) into the sinus node artery of the isolated atrium consistently caused slight negative chronotropic and rather marked negative inotropic effects.

3. After treatment with a relatively large dose of lignocaine, electrical stimulation‐induced negative chronotropic and inotropic responses were significantly inhibited in a dose‐related manner, but positive chronotropic and inotropic responses were slightly depressed only at an extremely high dose of lignocaine (10.0 μmol).

4. Noradrenaline‐induced positive chronotropic and inotropic effects were not modified by any doses of lignocaine used (0.3, 1.0, 3.0 and 10.0 μmol). Acetylcholine‐induced negative chronotropic and inotropic effects were slightly, but significantly, depressed by 10 μmol lignocaine.

5. These results suggest that a relatively large dose of lignocaine has a dominant presynaptic inhibitory action, particularly on the parasympathetic component.
Differential Secretion Of Adrenaline And Noradrenaline In Response To Various Secretagogues From Bovine Chromaffin Cells
Authors: Kuwashima Haruhiro , Matsumura Chiaki , Kimura Tomohiko
Published: March 2003

1. Differential secretion of adrenaline (Adr) and noradrenaline (NA) in response to various secretagogues was studied in bovine adrenal chromaffin cells.

2. Acetylcholine (ACh; 3–300 μmol/L), 1,1‐dimethyl‐ 4‐phenyl‐piperazinum (DMPP; 1–100 μmol/L), high K+ (20–60 mmol/L), calcimycin (1–100 μmol/L), histamine (0.3–30 μmol/L) and angiotensin (Ang)II (0.3–30 μmol/L) induced the secretion of a 1.3–2‐fold greater percentage of NA stores than Adr stores in intact cells.

3. In β‐escin‐permeabilized cells, Ca2+ (0.1–30 μmol/L) induced a greater secretion of Adr and NA in the presence of MgATP (2 mmol/L) than in the absence of MgATP. The percentage of NA secreted was 1.4‐ and 1.5‐fold greater than that of Adr in the presence and absence of MgATP, respectively.

4. The ATP‐independent phase of the Ca2+‐dependent exocytosis is thought to be associated with the final step that ultimately leads to fusion, while the ATP‐dependent phase is thought to be associated with the vesicle priming reaction. Therefore, the preferential secretion of NA in response to ACh, DMPP, high K+, calcimycin, histamine and AngII may be due, at least in part, to the greater effectiveness of Ca2+ in producing exocytosis in NA‐containing cells.
Quality Use Of Medicines: Where Does Pharmacology‘End’? Invited Ascept Lecture
Authors: Smith Aj
Published: March 2003

1. Pharmacology can be defined very narrowly (e.g. as solely a laboratory based specialty) or more broadly to include issues surrounding rational drug use in the community.

2. While the hypothetio–deductive approach is more clearly in evidence in laboratory and clinical pharmacological experimentation, it is no less important in the comparatively less‐well charted area of drug use in the community.

3. Just as laboratory pharmacology has built new partnerships with molecular biology and genetics, the exploration of community drug use brings pharmacologists into partnership with other disciplines (epidemiology, economics, behavioural science) and other sectors (consumers, government, health professionals and industry).

4. Implementing Australia’s Quality Use of Medicines Policy (1992) has led to vigorous exploration of issues and problems in medicinal drug use in the community and much scientific study of causes and potential interventions to improve the situation.

5. The Australian National Prescribing Service (1998) is attempting to bring an evidence‐based approach to the many facets of prescribing and drug use, by prescribers and consumers alike.

6. Innovation commonly occurs at the interface between the boundaries of traditional disciplines.
Tris Lipidation: A Chemically Flexible Technology For Modifying The Delivery Of Drugs and Genes
Authors: Lockett T , Reilly W , Manthey M , Wells X , Cameron F , Moghaddam M , Johnston J , Smith K , Francis C , Yang Q , Whittaker R
Published: March 2003

1. One of the major challenges in the development of pharmaceuticals is their formulation with other materials to give them the desired bioavailability profile when administered into the body.

2. We have developed a flexible platform technology (Tris lipidation) to simply and effectively alter the lipophilicity of drugs. As implied by the name, the technology uses the common buffer Tris as a linker between the drugs of interest and a domain of variable hydrophobicity.

3. We demonstrate, using a mouse melanoma model, that Tris‐lipidated conjugates of the widely used cytotoxic and anti‐inflammatory drug methotrexate (MTX) display enhanced potency in the local treatment of tumours and reduced systemic toxicity when compared with the unconjugated drug.

4. With genes now being predicted to be the pharmaceuticals of the future, we show that Tris‐lipidated cationic peptides can efficiently deliver DNA into (transfect) cells in culture. Furthermore, by comparing the abilities of variants of these Tris‐based cationic lipids to transfect cultured cells, we demonstrate that modifications made to variable regions of Tris‐lipidated compounds can dramatically alter their delivery profiles.
Modelling The Neural Control Of Intrarenal Blood Flow
Authors: Navakatikyan Michael A , Leonard Bridget L , Evans Roger G , Malpas Simon C
Published: March 2003

1. The aim of the present study was to produce a mathematical model that describes the way dynamic changes in renal sympathetic nerve activity affect renal, cortical and medullary blood flow.

2. Cortical blood flow (CBF) and medullary blood flow (MBF) were measured using laser‐Doppler flowmetry and (total) renal blood flow (RBF) was measured by transit‐time flowmetry in six pentobarbitone‐anaesthetized rabbits. The renal nerves were stimulated with rectangular pulses of 2 msec width and constant voltage at frequencies of 0.5, 1, 1.5, 2 and 3 Hz.

3. An exponential function with two parameters was applied; steady state gain and a dynamic constant for the blood flow reduction with stimulation. The steady state gain coefficients were similar for RBF and CBF, but significantly less for MBF. The time taken to reach minimum flow was less for MBF than for RBF and CBF.

4. The model parameters indicate that there is differential neural control of CBF and MBF.
Cytochrome P450‐Dependent Metabolites Of Arachidonic Acid And Renal Function In The Rat
Authors: Oyekan AO
Published: March 2003

1. The present study examined whether renal cytochrome P450 (CYP450)‐derived eicosanoids influence the pressure– natriuretic and haemodynamic responses to elevated renal perfusion pressure (RPP) in the rat.

2. Natriuresis and diuresis, as well as changes in renal blood flow (RBF) and glomerular filtration rate (GFR) following step‐wise elevations in RPP from 75 to 125 mmHg were compared in control rats and in rats treated with 12,12‐dibromodecenoic acid (DBDD; 2.5 mg/kg per h; n = 5), an inhibitor of ω/ω‐1 hydroxylase, or miconazole (1.3 mg/kg per h; n = 7), an inhibitor of epoxygenase.

3. In control rats, sodium excretion (UNaV) and urine volume (UV) increased five‐fold when RPP was increased from 75 to 125 mmHg, while RBF and GFR increased two‐fold when RPP increased from 75 to 100 mmHg, with no further increase between 100 and 125 mmHg, the autoregulatory range.

4. Miconazole, but not DBDD, altered the pressure– natriuresis relationship, exaggerating the increases in UNaV and UV three‐ to four‐fold when RPP was increased from 100 to 125 mmHg.

5. In contrast, DBDD eliminated the autoregulatory response because it abolished the plateau in RBF and GFR when RPP was increased from 100 to 125 mmHg, whereas miconazole was without effect.

6. These results suggest that CYP450‐dependent ω/ω‐1 hydroxylase metabolites of arachidonic acid contribute to vascular responses, while epoxygenase metabolites contribute to renal tubular responses to alterations in RPP in the rat.
Identification Of New Single‐Nucleotide Polymorphisms In The Thrombin Receptor Gene And Their Effects On Coronary Artery Diseases In Koreans
Authors: Park H‐Y , Nabika T , Jang Y , Kim D , Kim H‐S , Masuda J
Published: March 2003

1. The thrombin receptor (the protease‐activated receptor‐1; PAR‐1) is located on vascular cells as well as platelets and may play important roles in atherosclerotic disorders, such as coronary artery diseases (CAD). In the present study, we searched for genetic polymorphisms of the PAR‐1 gene and evaluated their effects on CAD by association analysis.

2. We identified six polymorphisms in the 5′‐untranslated region of the PAR‐1 gene by polymerase chain reaction–single‐strand conformation polymorphism (PCR‐SSCP); five single‐nucleotide polymorphisms (SNP) at –2355 (A to G), –2333 (T to G), –1428 (G to A), –1071 (C to T) and –561 (A to G) and a simple sequence repeat (SSR) polymorphism between –1935 and –1841. Five SNP were in strong linkage disequilibrium with each other to make three major haplotypes, the frequency of which was over 90% of all possible haplotypes.

3. For association analysis, 150 patients who had CAD (CAD+), 58 subjects who had no stenosis on the coronary angiogram and 186 reference subjects who had no clinical evidence of CAD were used from the Korean population. The genotype frequencies of the SNP were in Hardy–Weinberg equilibrium, except A‐561G in CAD+. The association of these SNP as well as of the SSR with CAD was not evident. This result suggests no major roles of the PAR‐1 gene in CAD in Koreans.
Cystic Fibrosis Transmembrane Conductance Regulator And The Outwardly Rectifying Chloride Channel: A Relationship Between Two Chloride Channels Expressed In Epithelial Cells
Authors: Hryciw Deanne H , Guggino William B
Published: March 2003

1. Mutations in the cystic fibrosis transmembrane conductance regulator (CFTR) result in the primary defect observed in patients with cystic fibrosis.

2. The CFTR is a member of the ATPase‐binding cassette (ABC) transporter family but, unlike other members of this group, CFTR conducts a chloride current that is activated by cAMP.

3. In epithelial cells, the cAMP‐stimulated chloride current is conducted by both CFTR and the outwardly rectifying chloride channel (ORCC).

4. The present review summarizes the current knowledge of the properties of the two channels, as well as their relationship. Because the gene encoding the ORCC has not been identified, a discussion as to possible candidates for this chloride channel is included.
Molecular Characteristics And Functional Diversity Of Clca Family Members
Authors: Pauli Bendicht U , Abdel‐Ghany Mossaad , Cheng Hung‐Chi , Gruber Achim D , Archibald Heather A , Elble Randolph C
Published: March 2003

1. In the present brief review, we describe some of the molecular and functional characteristics of a novel mammalian family of putative Ca2+‐activated chloride channels (CLCA).

2. So far, two bovine (bCLC1; bCLCA2 (Lu‐ECAM‐1)), three mouse (mCLCA1; mCLCA2; mCLCA3) and four human (hCLCA1; hCLCA2; hCLCA3; hCLCA4) CLCA family members have been cloned. Each CLCA exhibits a distinct, often overlapping, tissue expression pattern.

3. With the exception of the truncated secreted hCLCA3, all CLCA proteins are synthesized as an approximately 125 kDa precursor transmembrane glycoprotein that is rapidly cleaved into 90 and 35 kDa subunits.

4. The CLCA proteins expressed on the luminal surface of lung vascular endothelia (bCLCA2; mCLCA1; hCLCA2) serve as adhesion molecules for lung metastatic cancer cells, mediating vascular arrest and lung colonization.

5. Expression of hCLCA2 in normal mammary epithelium is consistently lost in human breast cancer and in all tumorigenic breast cancer cell lines. Re‐expression of hCLCA2 in human breast cancer cells abrogates invasiveness of Matrigel (BD Biosciences‐Labware, Bedford, MA, USA) in vitro and tumorigenicity in nude mice, implying that hCLCA2 acts as a tumour suppressor in breast cancer.
Bufokinin: Actions And Distribution In The Toad Cardiovascular System
Authors: Liu Lu , Shang Fei , Comis Alfio , Burcher Elizabeth
Published: March 2003

1. Bufokinin is a substance P‐like neuropeptide and potent spasmogen isolated from the intestine of the cane toad Bufo marinus. In the present study, we investigated the effects of bufokinin on systemic blood pressure and heart rate in the anaesthetized toad and the distribution of bufokinin‐like immunoreactivity in the toad vasculature.

2. Intravenous bufokinin caused a dose‐dependent fall in systemic blood pressure (maximum fall 20 mmHg) with an ED50 of 2.9 pmol. At higher doses, the effect was prolonged and blood pressure did not return to baseline within 60 min. There was no significant change in heart rate associated with hypotension.

3. Bufokinin‐like immunoreactivity was mapped in whole mounts of toad blood vessels and organs using a mouse polyclonal antibody BK3 (at 1 : 5000) and the avidin–biotin method. Bufokinin‐immunoreactive fibres were associated with most blood vessels examined: a moderately dense perivascular network of varicose fibres was present around renal arteries, with sparser immunoreactive fibres in the ventral aorta, sciatic artery, anterior abdominal vein and hepatic portal vein.

4. Bufokinin‐immunoreactive fibres, mainly following blood vessels, were seen in whole mounts of the urinary bladder and tongue, but not in the air sac. In the heart ventricle, varicose fibres were found in the valve cusps, intracardiac ganglia, epicardium and myocardium close to the endocardium, but not in the rest of the myocardium.

5. The vasodepressor action of bufokinin and the presence of bufokinin‐like immunoreactivity in varicose fibres in various vessels suggest a role for bufokinin in haemodynamic regulation and/or sensory nerve function in the toad. The lack of any reflex tachycardia in response to the falls in blood pressure was of note.
Proceedings of the Australian Neuroscience Society Symposium: Schizophrenia A PROPOSED PATHOLOGICAL MODEL IN THE HIPPOCAMPUS OF SUBJECTS WITH SCHIZOPHRENIA
Authors: Scarr E , Dean B
Published: March 2003

1. The hippocampal formation plays an important role in the normal functioning of the brain, being implicated in cognition and sensory gating, both of which are affected in schizophrenia. The hippocampal formation receives information from the association cortices, which is processed by glutamatergic transmission within the hippocampus. Dopamine, noradrenaline, 5‐hydroxytryptamine (5‐HT), acetylcholine and GABA, all of which have been proposed to play a role in the neurobiology of schizophrenia, can affect this transmission.

2. The advent of the ‘atypical’ antipsychotics, with their broad pharmacological spectra and improved therapeutic outcome, has revitalized research into neurotransmitter dysfunction other than that of dopamine. In particular, there has been interest in the serotonergic and cholinergic systems within the hippocampal formation because these are two of the transmitter systems targeted by clozapine and olanzapine.

3. From the study of these systems, using tissue obtained post‐mortem from subjects with schizophrenia, we propose that there is a hyperserotonergic state in the hippocampal formation of some subjects with schizophrenia caused by a conformational change in the 5‐HT transporter. The model we propose allows us to construct further studies that will test the consequences of such a hyperserotonergic state in the hippocampal formation. This model has the potential to open new avenues in schizophrenia research.
Effects Of Ageing On The Activation Metabolism Of Rat Papillary Muscles
Authors: Kiriazis Helen , Gibbs Colin L
Published: March 2003

1. A myothermic technique has been used to investigate the mechanics and energetics of left ventricular papillary muscles from 6‐, 15‐, 22‐ and 27–32‐month‐old Sprague‐Dawley rats.

2. There was a significant increase in the left ventricular mass to body mass (LVM:BM) ratio in the senescent, 27–32‐month‐old group of animals compared with the younger animals (P < 0.05).

3. The maximum stress developed in the senescent groups was reduced by almost 40% in comparison with the stress developed by the 6‐, 15‐ and 22‐month‐old groups (P < 0.001). The mean rise time, half relaxation time and half width were increased significantly (P < 0.05) in the 22‐month‐old group but, unexpectedly, this effect was not seen in the senescent group.

4. Heat production per beat versus total stress relationships were obtained in two different ways to determine the magnitude of the activation heat and the isometric economy (given by the slope of the relationship). The activation heat was not significantly different between groups with either method, but there was a significant increase (P < 0.001) in the economy with which stress was developed in the senescent group in comparison with the 6‐ and 15‐month‐old groups.

5. A combination of forskolin (2.5–6.5 μmol/L) and high Ca2+ (7.5 mmol/L) was used to increase the energy usage per beat. In the 6‐ and 15‐month‐old groups, these agents caused a four‐fold increase in the activation heat magnitude compared with a less than two‐fold increase in the 22‐month‐old and senescent groups (P < 0.001). There was no effect of forskolin/high calcium on the slope of the heat : total stress relationship.

6. The data suggest that, under conditions known to increase cardiac contractility, there is a reduced ability to cycle calcium in the 22‐month‐old and senescent groups relative to the young adult 6‐month‐old and adult 15‐month‐old groups.
Two Possible Mechanisms Underlying Nitrate Tolerance In Monkey Coronary Arteries
Authors: Omura Tomoko , Matsumoto Tetsuya , Nakae Ichiro , Takahashi Masayuki , Kinoshita Masahiko
Published: March 2003

1. Previous studies using isolated arteries have demonstrated cross‐tolerance between nitric oxide (NO) donors such as nitroglycerin (NTG) and sodium nitroprusside (SNP). However, it remains unclear whether the vasorelaxing effect of atrial natriuretic peptide (ANP), an activator of particulate guanylate cyclase, is affected by treatment with NO donors. To investigate the cross‐tolerance and interactions between NTG and ANP in coronary vasorelaxant responses, we used two models of monkey coronary arterial strips (Macaca fuscata).

2. In one model, which was induced by a 1 h treatment with 4.4 × 10–4 mol/L NTG followed by washout of the agent for 1 h, the vasorelaxing effects of subsequent NTG were markedly attenuated, whereas those of ANP and NO were not affected. These findings suggest that the development of NTG tolerance is associated with a biotransformation process from NTG to NO. In the other model, which did not include washout after exposure to 3 × 10–6 mol/L NTG, the vasorelaxant responses to 10–8 mol/L ANP (31.1±5.4 vs 5.1±2.1%, respectively; P < 0.001), 10–6 mol/L NO (61.5±2.4 vs 29.5±8.5%, respectively; P < 0.001) and 10–8 mol/L SNP (49.4±6.4 vs 8.0±2.0%, respectively; P < 0.001) were significantly attenuated. The concentration– response curve for 8‐bromo‐cGMP (8‐Br‐cGMP) was shifted to the right, whereas responses to papaverine and forskolin were unchanged. These findings suggest that an intracellular process that occurs after the synthesis of cGMP is responsible for this interaction.

3. As a mechanism of NTG tolerance, two possible processes may be impaired: (i) biotransformation from NTG to NO; and (ii) an intracellular process that occurs after the synthesis of cGMP.
Gene Expression Studies Using Microarrays
Authors: Burgess Janette K
Published: March 2003

1. The rapid progression of the collaborative sequencing programmes that are unravelling the complete genome sequences of many organisms are opening pathways for new approaches to gene analysis. As the sequence data become available, the bottleneck in biological research will shift to understanding the role of each of these genes. Techniques that enable the analysis of large sets of genes in one experiment will elucidate the interactions of genes in diverse biological systems.

2. Microarrays, which consist of large numbers of cDNA or oligonucleotides spotted onto a glass microscope slide, are one such technology. RNA isolated from two populations of cells, one control and one altered by experimental treatment or disease, is labelled with two different fluorochromes before being hybridized to the microarray. After a standard hybridization reaction, a scanner records the intensity of the two fluorochromes. The data can be analysed using special software that enables clustering of genes that have similar expression patterns.

3. Such powerful analysis techniques will provide information about genes whose functions are currently unknown and enhance our understanding of how genes interact to provide molecular control. This increase in knowledge about gene function will allow new targeted approaches for the development of drugs and/or gene therapies.
Extracellular ATP and cAMP as Paracrine and Interorgan Regulators of Renal Function P2Y Receptors of MDCK Cells: Epithelial Cell Regulation by Extracellular Nucleotides
Authors: Insel Paul A , Ostrom Rennolds S , Zambon Alexander C , Hughes Richard J , Balboa Maria A , Shehnaz Darakhshanda , Gregorian Caroline , Torres Brian , Firestein Bonnie L , Xing Mingzhao , Post¥ Steven R
Published: March 2003

1. Madin–Darby canine kidney (MDCK) cells, a well‐ differentiated renal epithelial cell line derived from distal tubule/collecting duct, respond to extracellular nucleotides by altering ion flux and the production of arachidonic acid‐derived products, in particular prostaglandin E2 (PGE2). Our work has defined the receptors and signalling events involved in such responses.

2. We have found evidence for expression of at least three P2Y receptor subtypes (P2Y1, P2Y2 and P2Y11) in MDCK‐D1 cells, a subclone from parental MDCK.

3. These receptors appear to couple to increases in calcium and protein kinase C activity, probably via a Gq/G11‐mediated activation of phospholipase C.

4. In addition, P2Y receptor activation can promote a prominent increase in cAMP. This includes both a P2Y2 receptor‐mediated cyclo‐oxygenase (COX)‐dependent component and another COX‐independent component mediated by other P2Y receptors.

5. We have documented that changing media in which cells are grown releases ATP and, in turn, activates P2Y receptors. Such release of ATP contributes in a major way to basal cAMP levels in these cells.

6. The data indicate that MDCK cells are a useful model to define the regulation of epithelial cells by extracellular nucleotides. Of particular note, spontaneous or stretch‐induced release of ATP and subsequent activation of one or more P2Y receptors contributes to establishing the basal activity of signalling pathways.
John Ludbrook APPS Symposium Statistics In Physiology And Pharmacology: A Slow And Erratic Learning Curve
Authors: Ludbrook John
Published: March 2003

1. Learning how to apply statistical analyses to the results of experimental or clinical studies may take a lifetime of trial (and sometimes error), as it has done in the author’s case. There is no evidence that biomedical investigators of the present generation are on a steeper learning curve. Gross misunderstandings of the purpose and functions of statistical analysis are apparent in applications to research grant‐giving bodies and ethics committees, in manuscripts submitted to journals and sometimes in published papers.

2. Although estimation of minimal group (sample) size for a given power is an essential step in planning clinical studies, it seems to be used rarely in laboratory experimental work. This is despite exhortations to restrict the number of animals used to a minimum.

3. Most investigators use hypothesis testing to analyse their results, but their understanding of the meaning of the resultant P‐values is slight.

4. A flaw found almost universally in biomedical manuscripts is to make multiple inferences from the results of a single study. The goal of statistical analysis is to maintain the familywise type I error rate (risk of false‐positive inference) at a predetermined level (usually 5%). But, when multiple inferences are made from the same experiment, the risk of false‐positive error is inflated. There are two solutions to this problem: (i) use a multiple comparison procedure to control the familywise type I error rate; and (ii) test a single, global hypothesis.

5. Biomedical investigators have been quick to acquire computer statistics software and to use it to analyse their experiments. However, they have been slow to recognize the limitations of this software. These include: (i) inadequate documentation of routines, so that neither the user nor the reader of published papers can be sure how the tests have been executed; (ii) flawed algorithms for the execution of statistical procedures; and (iii) failure to recognize that the best software for their purposes is that which takes them just beyond their statistical horizons.

6. The obvious solution to these difficulties is to recruit a biomedical statistician into every research group, at a relatively trivial cost. However, properly qualified biostatisticians are in desperately short supply in Australia. It follows that research groups, national grant‐giving agencies and academic institutions must make provision for the proper training and subsequent employment of biostatisticians.
Circumventricular Organs: Gateways to the Brain Axonal Projections From The Organum Vasculosum Lamina Terminalis To The Supraoptic Nucleus: Functional Analysis And Presynaptic Modulation
Authors: Bourque Charles W , Richard Dominique
Published: March 2003

1. The rat organum vasculosum lamina terminalis (OVLT) contains GABA‐ and glutamate‐releasing neurons that project directly to magnocellular neurosecretory cells (MNC) in the supraoptic nucleus.

2. Changes in osmolality over the OVLT in hypothalamic explants cause proportional changes in firing in MNC through corresponding changes in the frequency of spontaneous glutamatergic excitatory post‐synaptic potentials without affecting GABAergic inhibitory post‐synaptic potentials.

3. Exogenously applied atrial natriuretic peptide inhibits the osmotic control of MNC by causing a decrease in the amount of glutamate released provoked by action potentials originating from OVLT neurons.
Effects of dopamine d2 receptor agonists in a pituitary transplantation‐induced hyperprolactinaemia/anovulation model in rats
Authors: Moro Makoto , Inada Yoichi , Miyata Hiroshi , Komatsu Hidetada , Kojima Masami , Tsujii Hirotada
Published: March 2003

1. In the present study, we investigated the effects of hyperprolactinaemia, induced by transplantation of anterior pituitary glands under the kidney capsule in female rats, on the relationship between serum and pituitary concentrations of the gonadotropins and on the oestrous cycle.

2. Rats with pituitary transplants showed increased serum prolactin concentrations and decreased serum concentrations of gonadotropins and increased pituitary concentrations of gonadotropins. Moreover, these rats showed persistent dioestrous and anovulation from 3 to 6 days after transplantation.

3. A single oral administration of cabergoline (at doses between 0.001 and 0.1 mg/kg) dose‐dependently inhibited the elevated serum prolactin concentrations in hyperprolactinaemic rats. At 0.1 mg/kg, cabergoline induced a continuous reduction in serum prolactin concentrations for 5 days after administration. Terguride (0.1 mg/kg) and bromocriptine (10 mg/kg) also reduced serum prolactin concentrations at 1 and 3 days after administration. All three dopamine D2 receptor agonists increased serum gonadotropin concentrations and ovarian weight at 3 days after administration.

4. In rats exhibiting anovulation, a single oral administration of any one of the three dopamine D2 receptor agonists dose‐dependently restored ovulation and a normal oestrous cycle appeared. Oral administration of cabergoline (0.03 mg/kg) or terguride (0.1 mg/kg) restored ovarian function and abolished the anovulation following a reduction in serum prolactin concentrations. However, bromocriptine (10 mg/kg) did not completely abolish anovulation. Following administration of terguride (0.3 mg/kg) or bromocriptine (30 mg/kg), only one normal oestrous cycle appeared; however, following cabergoline (0.1 mg/kg), two normal oestrous cycles appeared.

5. These results suggest that cabergoline has a potent and long‐lasting action as a dopamine D2 receptor agonist and, thus, should be a useful drug for the treatment of galactorrhoea and hyperprolactinaemic amenorrhoea and/or anovulation in humans.
Coronary Artery Baroreceptor‐Mediated Changes In Arterial Pressure: A Pilot Study In Conscious And Anaesthetized Sheep
Authors: Bennetts JS , Arnolda LF , Cullen HC , Knight JL , Baker RA , McKitrick DJ
Published: March 2003

1. Evidence suggesting the presence of coronary artery baroreceptors on coronary arteries has existed for over 30 years.

2. Evidence that activation of ventricular mechanoreceptors can elicit cardiovascular changes has been challenged, with those changes now thought to be due to coronary artery mechanoreceptors.

3. Studies have suggested that coronary artery mechanoreceptors act as coronary baroreceptors with a role in cardiovascular regulation. However, all evidence to date has been obtained in anaesthetized animal models in physiologically compromised intra‐operative states.

4. The purpose of the present study was to design an ovine model that would allow the discrete stretch of coronary arteries without causing ischaemia or changing flow or intra‐arterial pressure and that would confirm results seen in previous studies. In addition, the possibility that the technique could be used for studies of coronary artery baroreflexes in conscious sheep was investigated.

5. Controlled stretch of the proximal left anterior descending coronary artery elicited decreases in arterial pressure without changes in heart rate or electrocardiographic activity in halothane‐anaesthetized sheep. Similar results were demonstrated in conscious sheep after surgical recovery of up to 2 weeks.

6. The present study supports the possibility that coronary artery baroreceptors exist and likely have a role in cardiovascular regulation. The results of the present study in anaesthetized sheep are in agreement with previous results in anaesthetized animals, but also provide the first demonstration of coronary baroreceptor activity in a conscious animal model, underscoring the potential use of the model in the study of coronary artery baroreceptors in the intact animal.
Vasomotor Effects Of ARG–Gly–ASP (RGD) Peptides Are Limited And Not Related To Endothelium‐Derived Hyperpolarizing Factor‐Mediated Relaxation In Rat Mesenteric Arteries
Authors: Bakker Erik Ntp , Balt Jippe C , Pfaffendorf Martin , Spaan Jos Ae , VanBavel Ed
Published: March 2003

1. In the present study we tested the effect of arg–gly–asp (RGD) peptides on vasomotor responses in rat isolated mesenteric arteries. More specifically, the hypothesis was tested that RGD interaction with integrins mediates relaxation attributed to endothelium‐derived hyperpolarizing factor (EDHF).

2. The presence of the β3 integrin subunit was shown by western blot analysis. To study its functional role, arteries (355 ± 11 μm; n = 50) were mounted in a wire myograph set‐up to measure isometric force generation. After blockade of nitric oxide synthesis with NG‐nitro‐L‐arginine (0.1 mmol/L) and prostaglandin synthesis with indomethacin (10 μmol/L), methacholine (10 μmol/L) induced a transient relaxation within 1 min of 72 ± 4.0% (as percentage of precontraction with phenylephrine; n = 27).

3. These responses were inhibited by a 60 mmol/L potassium buffer (18 ± 6.0%; n = 6) or endothelium denudation (12 ± 3.2%; n = 7), consistent with EDHF.

4. A function‐blocking monoclonal antibody against the integrin β3 chain did not affect relaxation.

5. The RGD peptides gly–arg–gly–asp–thr–pro (GRGDTP), gly–arg–gly–asp–ser (GRGDS) and cyclic RGD, ligands for the RGD binding site of integrins, also did not affect relaxation induced by methacholine.

6. Cyclic RGD increased contraction from 91 ± 3 to 98 ± 3% (as percentage of 120 mmol/L potassium).

7. In conclusion, these data show that vasomotor responses related to integrins are small and not involved in hyperpolarization attributed to EDHF in rat mesenteric artery.
An Overview Of New And Novel Pharmacotherapies For Use In Traumatic Brain Injury
Authors: Vink Robert , Nimmo Alan J , Cernak Ibolja
Published: March 2003

1. Although a number of interventional pharmacotherapies have undergone clinical trial in traumatic brain injury (TBI), none has shown considerable promise. The present short review will examine some of the more novel compounds that have been proposed recently as potential therapeutic agents for use in TBI.

2. Previous experimental studies have demonstrated that brain intracellular free magnesium significantly declines following TBI and that the administration of magnesium salts attenuates the post‐traumatic neurological deficits. More recent studies have established that magnesium salts administered after trauma enter the brain intracellular space and reduce the size of the lesion volume. Such protection could be afforded through attenuation of both necrotic and apoptotic cell death. Magnesium salts are currently on clinical trial in TBI.

3. Cyclosporine A is known to inhibit opening of the mitochondrial permeability transition pore. Administration of cyclosporine A after TBI has been shown to attenuate axonal injury and decrease the resultant lesion volume. Therefore, inhibitors of mitochondrial transition pore opening and resultant attenuation of apoptosis show some promise as neuroprotective agents.

4. Recent evidence has shown that substance P antagonists may decrease lesion volume and improve neurological outcome after ischaemia. Similar findings have recently been reported in TBI. The fact that substance P antagonists are known to reduce neurogenic inflammation, oedema formation and are clinically being trialed as both antidepressants and antinociceptive agents suggests that these agents warrant further investigation as therapeutic agents following TBI.

5. There are numerous contradictions in the literature regarding the potential neuroprotective effects of the hormones oestrogen and progesterone. Recent studies suggest that both hormones are protective in TBI and further studies are required to ascertain the mechanisms associated with this protection and any potential for clinical application.
Activation Of Cyclo‐Oxygenase‐2 Contributes To Motor And Cognitive Dysfunction Following Diffuse Traumatic Brain Injury In Rats
Authors: Cernak I , O'Connor C , Vink R
Published: March 2003

1. Post‐traumatic inflammation may play a significant role in the development of delayed secondary brain damage following traumatic brain injury.

2. During post‐traumatic inflammation, metabolic products of arachidonic acid, known as prostanoids (prostaglandins and thromboxanes) are released and aggravate the injury process. Prostanoid synthesis is regulated by the enzyme cyclo‐oxygenase (COX), which is present in at least two isoforms, COX‐1 (the constitutive form) and COX‐2 (the inducible form).

3. In the present study, we examine the temporal and spatial profiles of COX‐2 expression and the effects of the COX‐2 inhibitor nimesulide on motor and cognitive outcome following diffuse traumatic brain injury in rats.

4. Adult male Sprague‐Dawley rats were injured using the 2 m impact acceleration model of diffuse traumatic brain injury. At preselected time points after injury, animals were killed and the expression of COX‐2 was measured in the cortex and hippocampus by western blotting techniques.

5. Increased expression of COX‐2 was found in the cortex at 3 days and in the hippocampus as early as 3 h postinjury and this persisted for at least 12 days.

6. Administration of nimesulide (6 mg/kg, i.p.) at 30 min after injury and daily over a 10 day post‐traumatic neurological assessment period resulted in a significant improvement compared with vehicle (2% dimethylsulphoxide diluted in isotonic saline)‐treated controls in cognitive deficits, as assessed by the Barnes circular maze. There was also a significant improvement in motor dysfunction as assessed by the rotarod test on days 1 and 2 post‐trauma compared with vehicle‐treated controls.

7. These results implicate the involvement of COX‐2 in cognitive and motor dysfunction following diffuse traumatic brain injury.
Neural Pathways From The Lamina Terminalis Influencing Cardiovascular And Body Fluid Homeostasis
Authors: McKinley MJ , Allen AM , May CN , McAllen RM , Oldfield BJ , Sly D , Mendelsohn FAO
Published: March 2003

1. The lamina terminalis, a region of the brain with a high concentration of angiotensin AT1 receptors, consists of three distinct nuclei, the median preoptic nucleus, the subfornical organ and organum vasculosum of the lamina terminalis (OVLT). These latter two regions lack a blood–brain and detect changes in plasma angiotensin (Ang) II concentration and osmolality.

2. Efferent neural pathways from the lamina terminalis to the hypothalamic paraventricular and supraoptic nuclei mediate vasopressin secretion in response to plasma hypertonicity and increased circulating levels of AngII.

3. Studies using the neurotropic virus pseudorabies, which undergoes retrograde transynaptic neuronal transport following injection into peripheral sites, show that neurons in the lamina terminalis have efferent polysynaptic neural connections to the peripheral sympathetic nervous system. Some of these neurons have been shown to have polysynaptic connections to the kidney and to express AT1 receptor mRNA. We propose that circulating AngII acts at AT1 receptors in the subfornical organ and OVLT to influence the sympathetic nervous system. It is likely that the neural pathway subserving this influence involves a synapse in the hypothalamic paraventricular nucleus.

4. The lamina terminalis may exert an inhibitory osmoregulatory influence on renin secretion by the kidney. This osmoregulatory influence may be mediated by inhibition of renal sympathetic nerve activity and appears to involve a central angiotensinergic synapse.

5. The lamina terminalis exerts an osmoregulatory influence on renal sodium excretion that is independent of the renal nerves and is probably hormonally mediated.
Heterogeneity Of The Properties Of INa in Epicardial And Endocardial Cells Of Rat Ventricle
Authors: Honen Bonny N , Saint David A
Published: March 2003

1. Ventricular INa heterogeneity was investigated in adult rat hearts.
Differences in transient outward potassium current (Ito) were used to
confirm isolation of subepicardial and subendocardial cells. Mean peak Ito
was 6.0 ± 0.7 and 1.6 ± 0.45 pA/pF in epicardial and
endocardial cells, respectively (P < < 0.01).

2. Maximum sodium conductance was smaller in subendocardial cells compared with subepicardial cells (2.39 ± 0.11 vs 2.78 ± 0.12
nS/pF, respectively; n = 17 for both; 0.01 < P < 0.05) and 50%
activation occurred at a slightly more negative potential (–47.6 ±
0.8 vs–44.9 ± 0.9 mV, respectively; n = 10 for
both; 0.01 < P < 0.05).

3. The potential for 50% inactivation was not significantly different in subepicardial compared with subendocardial cells (72.2 ± 1.0 vs
72.8 ± 2.2 mV, respectively; n = 17 for both; NS).

4. Persistent sodium current density appeared smaller in subendocardial (n
= 19) compared with subepicardial (n = 11) cells (at a test potential of –25
mV current, density was 0.118 ± 0.041 vs 0.144 ± 0.085 pA/pF, respectively), although this was not statistically significant due to large variability between cells.

5. Mathematical modelling of the cardiac action potential indicated that the combined effects of differences in current density and voltage dependence of sodium currents are unlikely to contribute to ventricular action potential heterogeneity between epicardial and endocardial cells.
Val‐Tyr As A Natural Antihypertensive Dipeptide Can Be Absorbed Into The Human Circulatory Blood System
Authors: Matsui Toshiro , Tamaya Kei , Seki Eiji , Osajima Katsuhiro , Matsumoto Kiyoshi , Kawasaki Terukazu
Published: March 2003

1. Intact absorption of the bioactive dipeptide Val‐Tyr (VY), with in vivo antihypertensive ability in normotensive human subjects, was investigated.

2. As a result of a single oral administration of VY, the VY absorption curve occurred maximally over the second hour postprandially; a greater than 10‐fold higher increment of VY following a dose of 12 mg was observed in the plasma at 2 h compared with the baseline concentration of VY at 0 h (1934 ± 145 vs 159 ± 11 fmol/mL plasma, respectively).

3. Plasma VY levels increased with dose administered (3, 6 and 12 mg), suggesting that exogenous VY could be absorbed intact into the human blood depending on the dose. The elimination half time (t1/2)
of VY was estimated to be 3.1 h. The area under the curve for the 12 mg VY dose was
9185 ± 688 fmol·h/mL plasma.