Showing papers in "Experimental Physiology in 1997"

Oxidative stress: oxidants and antioxidants

Abstract: An imbalance between oxidants and antioxidants in favour of the oxidants, potentially leading to damage, is termed 'oxidative stress'. Oxidants are formed as a normal product of aerobic metabolism but can be produced at elevated rates under pathophysiological conditions. Antioxidant defense involves several strategies, both enzymatic and non-enzymatic. In the lipid phase, tocopherols and carotenes as well as oxy-carotenoids are of interest, as are vitamin A and ubiquinols. In the aqueous phase, there are ascorbate, glutathione and other compounds. In addition to the cytosol, the nuclear and mitochondrial matrices and extracellular fluids are protected. Overall, these low molecular mass antioxidant molecules add significantly to the defense provided by the enzymes superoxide dismutase, catalase and glutathione peroxidases.

Starling: the formulation of his hypothesis of microvascular fluid exchange and its significance after 100 years.

Abstract: Introduction 1 Starling, the man 2 The formulation of Starling's hypothesis 6 Lymph formation 6 Filtration and reabsorption: Starling puts forward his hypothesis 9 Reactions to Starling's hypothesis 11 Starling's hypothesis and Starling's principle 14 Hydrostatic and oncotic pressure of the interstitial fluid 15 Fluid exchange in single capillaries 17 Starling pressures and steady-state fluid exchange 21 Starling pressures in dependent regions of the body 23 Steady reabsorption of fluid in specialized tissues 25 Conclusions 27 References 27

Role of nuclear factor‐kappa B in atherogenesis

Abstract: Transcription factors of the nuclear factor-kappa B (NF-kappa B)/Rel family have an important function in the regulation of a variety of genes involved in the inflammatory and proliferative responses of cells. Recent studies strongly indicate that the inducible transcription factor NF-kappa B is involved in the pathogenesis of atherosclerosis. Activated NF-kappa B is present in the fibrotic thickened intima-media and atheromatous areas of the atherosclerotic lesion, within smooth muscle cells, macrophages and endothelial cells, whereas little or no activated NF-kappa B can be detected in vessels lacking atherosclerosis. A variety of molecules have been identified in the atherosclerotic environment that are able to activate NF-kappa B in vitro. Furthermore, an increased expression of numerous genes known to be regulated by NF-kappa B has been found in the atherosclerotic lesion. Possible functional implications for activated NF-kappa B in atherogenesis are discussed here. The activation and role of NF-kappa B in atherosclerosis may provide a model for the involvement of the transcription factor in human chronic inflammatory disease.

Programmed cell death in bovine mammary tissue during lactation and involution.

Abstract: Cessation of milk removal causes mammary tissue involution, which in rodents is characterized by extensive tissue degeneration and loss of the majority of luminal epithelial cells by apoptosis. In contrast, bovine mammary tissue shows little histological evidence of tissue remodelling between lactations. In this study, we combined histology with molecular biology to examine the cellular and molecular changes in bovine mammary tissue on cessation of milking. Oligonucleosomal laddering of genomic DNA extracted from lactating tissue indicated that a proportion of cells were dying by apoptosis. This was confirmed by terminal deoxynucleotide transferase-mediated deoxyuridine nick end-labelling of apoptotic cells in lactating tissue sections (TUNEL). One week after cessation of milking, alpha-lactalbumin and alpha S1-casein messenger RNA (mRNA) abundance had decreased by 99 and 85%, respectively, whereas lactoferrin mRNA had increased 20-fold. Drying off was also accompanied by an increase in oligonucleosomal laddering of genomic DNA, and by an increase in the proportion of TUNEL-positive cells, which were localized preferentially in regions where alveolar structure had deteriorated. Therefore, termination of lactation was associated with partial loss of the mammary cell population and dedifferentiation of the remainder.

Dysfunction of the endothelial nitric oxide signalling pathway in diabetes and hyperglycaemia

Abstract: 1. Vasodilatation induced by adenosine is mediated through the activation of A2-purinoceptors in endothelial and smooth muscle cells. 2. Adenosine induces a rapid and transient membrane hyperpolarization in endothelial cells. 3. Acute exposure to adenosine or A2-purinoceptor agonists activates the L-arginine-NO signalling pathway in human endothelial cells in vitro.

Placental transporter activity and expression in relation to fetal growth

Abstract: The question of whether there are causative or compensatory changes in placental transport physiology affecting fetal growth is considered. Reductions in uterine and umbilical blood flow in growth retardation will reduce maternofetal exchange of lipophilic solutes, such as O2 and CO2, but will not have a major effect on the transfer of hydrophilic solutes. These solutes are transferred across the placenta by paracellular diffusion, transporter protein-mediated transport and endocytosis-exocytosis. Neither paracellular diffusion nor endocytosis-exocytosis has been investigated in relation to fetal growth. The weight of evidence is that there is no change in the activity and expression of the syncytiotrophoblast GI UTI glucose transporter in fetal growth retardation. However, there is strong evidence that the activity of the system A amino acid transporter, per milligram of placental membrane protein, is altered in relation to fetal growth, but in a complex manner. There is also some weaker evidence that the activity of the Na(+)-H+ exchanger, per milligram of placental membrane protein, is directly related to birth-weight. There are no data for other solute transporters; a considerable amount of work still remains to be done in order to understand the relationship between placental function and fetal growth rate.

The control of blood flow to the placenta

Abstract: The maintenance of adequate blood flow to the placenta is essential for the successful outcome of pregnancy. The placental vascular bed is often regarded as a low-resistance circulation in which blood flow is determined by the fetal cardiac output, but in pregnancies associated with growth retardation, and for reasons unknown, vascular resistance increases and blood flow is compromised. This review concentrates on recent advances in our understanding of the factors that may influence fetoplacental blood flow, with particular emphasis upon the circulation of the human placenta. The placenta has no neuronal input and vascular resistance is entirely determined by humoral and structural factors. In the absence of catecholamine sensitivity, constrictor prostanoids and endothelin may be important constrictor agonists, whereas nitric oxide is now considered to contribute to tonic reduction in vascular resistance. Little is known of the relative contributions of these vasoactive agents in the elevation of fetoplacental vascular resistance, but recent evidence points to an important role for altered vascular structure in growth-retarded pregnancies.

Cardiovascular and pancreatic endocrine responses to glucagon‐like peptide‐1(7–36) amide in the conscious calf

Abstract: Intravenous infusions of glucagon-like peptide-1(7-36) amide (GLP-1; 35 pmol min-1 kg-1 for 10 min) produced a significant rise in mean heart rate, without significant change in mean aortic blood pressure, together with a significant rise in mean arterial plasma insulin, but not in plasma pancreatic glucagon or pancreatic polypeptide concentration, in conscious calves given exogenous glucose (30-60 micromol min-1 kg-1 i.v.). The insulinotropic effect was eliminated in the presence of exogenous amino acids (0.03 mmol min-1 kg-1 i.v.). It was not affected predictably by blocking the synthesis of nitric oxide or by the simultaneous administration of the established incretin factor gastrin-releasing peptide (GRP). Whereas GLP-1 produced a statistically significant rise in plasma insulin concentration in these animals, it was much less effective than GRP in this respect, when given by continuous i.v. infusion.

Adipose tissue development during early postnatal life in ewe-reared lambs.

Abstract: This study examines the precise time course that brown adipose tissue (BAT) takes to adopt the characteristics of white adipose tissue in postnatal lambs. Perirenal adipose tissue was sampled from ewe-reared lambs within 1 h of birth and at 1, 2, 4, 7, 14, 21 and 30 days of age and analysed for the amount of mRNA for uncoupling protein (UCP), the amount and activity of UCP, and protein, mitochondrial protein and lipid content. This was combined with measurements of colonic temperature and jugular venous plasma concentrations of thyroid hormones and insulin-like growth factor-1 (IGF-1). Over the first 4-7 days of age, large quantities of UCP mRNA were associated with a peak in plasma triiodothyronine concentration at 2 days of age followed by a maximal amount and activity of UCP at 4 days and a basal colonic temperature of 39.3 degrees C. Between 7 and 30 days there was a large increase in lipid deposition as the amount and activity of UCP and the amount of UCP mRNA declined to basal values and colonic temperature was maintained at 40 degrees C. A significant positive relationship between perirenal adipose tissue lipid content and plasma IGF-1 concentration was observed throughout the study period. It is concluded that ovine adipose tissue maturation occurs in two distinct phases over the first month of life. The precise time scale of this process could be regulated in part by the lamb's body temperature which determines whether adipose tissue is required for heat production (i.e. BAT) or as an endogenous energy source (i.e. white adipose tissue).

Endothelial barrier dysfunction and oxidative stress: roles for nitric oxide?

Abstract: Endothelial dysfunction has an important role to play in the pathophysiology of human vascular disease. The maintenance of barrier function is critical to the role of vascular endothelium in cardiovascular haemostasis and this function can be compromised by inflammatory mediators, cytokines or oxidants. Under conditions of oxidative stress a variety of reactive oxygen species (ROS) may be generated, which increase the permeability of the endothelial monolayer to fluid, macromolecules and inflammatory cells. The endothelium-derived nitric oxide radical (NO), whose physiological actions include effects on vascular smooth muscle, is normally inactivated by the superoxide radical anion. While large amounts of NO have cytotoxic potential, it is now becoming clear that combinations of NO with ROS can produce either cytotoxic or cytoprotective effects, depending on the relative amounts of each which are present in the target cell or its environment at a particular time. The contribution of NO to oxidant-mediated endothelial barrier dysfunction can be assessed in vitro in endothelial monolayers grown on porous membrane supports. In this model, using hydrogen peroxide (H2O2) as the oxidant, H2O2-induced losses of barrier function can be enhanced or partially offset by NO donor drugs, depending on the concentration of NO donor used. Furthermore, the injurious or cytoprotective effects of these agents appear to be determined by the quantity of NO generated. Since NO is administered clinically by inhalation in conditions such as pulmonary hypertension or the adult respiratory distress syndrome, which are themselves associated with generation of ROS, it is likely that low concentrations of NO may protect the pulmonary vascular endothelium while high concentrations might be expected to combine with ROS to yield intermediates capable of causing further endothelial injury or loss of barrier function.

Engine and radiator: fetal and placental interactions for heat dissipation

Abstract: The 'engine' of fetal metabolism generates heat (3-4 W kg-1 in fetal sheep) which has to be dissipated to the maternal organism Fetal heat may move through the amniotic/allantoic fluids to the uterine wall (conductive pathway; total conductance, 11 W degrees C-1 kg-1) and with the umbilical arterial blood flow (convective pathway) to the placenta Because resistance to heat flow is larger than zero fetal temperature exceeds maternal temperature by about 05 degree C (03-1 degree C) Probably 85% of fetal heat is lost to the maternal organism through the placenta, which thus serves as the main 'radiator' Placental heat conductivity appears to be extremely high and this may lead to impaired heat exchange (guinea-pig placenta) A computer simulation demonstrates that fetal temperature is essentially clamped to maternal temperature, and that fetal thermoregulatory efforts to gain thermal independence would be futile Indeed, when the late gestational fetus in utero is challenged by cold stress, direct and indirect indicators of (non-shivering) thermogenesis (oxygen consumption, increase of plasma glycerol and free fatty acid levels) change only moderately In prematurely delivered lambs, however, cold stress provokes summit metabolism and maximum heat production Only when birth is imitated in utero (by cord clamping, external artificial lung ventilation and cooling) do thermogenic efforts approach levels typical of extra-uterine life This suggests the presence of inhibitors of thermogenesis of placental origin, eg prostaglandins and adenosine When the synthesis of prostaglandins is blocked by pretreatment with indomethacin, sheep fetuses react to intra-uterine cooling with vigorous thermogenic responses, which can be subdued by infusion of prostaglandin E2 (PGE2) Since the sheep placenta is known to produce sufficient amounts of PGE2, it seems that the placenta controls fetal thermogenic responses to some extent This transforms the fetus into an ectothermic organism, and yet allows the newborn the full exploitation of thermoregulatory responses typical of endothermic animals

The Wellcome Prize Lecture. Calcium entry mechanisms in human platelets

Abstract: Platelets play an essential role in haemostasis. Resting platelets circulate as biconcave discs, just 3,um in diameter and 1 um thick. At sites of vascular damage they are activated by multiple factors to undergo a dramatic transformation, becoming more spherical, throwing out pseudopodia and exposing fibrinogen receptors as a haemostatic plug starts to form (for reviews see Siess, 1989; Sargeant & Sage, 1994). This plug then grows and is reinforced as the platelets themselves secrete cross-linking proteins and platelet agonists, including ADP, ATP and thromboxane A2' Activation also results in the loss of plasma membrane phospholipid asymmetry and the shedding of microvesicles. The resulting exposure of phosphatidylserine and other negatively charged phospholipids in the outer membrane leaflet forms a procoagulant surface essential for the generation of several clotting factors, including the most potent platelet agonist, thrombin. A rise in cytosolic calcium concentration ([Ca2+]i) is a major component of the intracellular stimulus to these events, all of which can be evoked if [Ca2+]i is sufficiently raised using a Ca2+ ionophore. Physiologically, however, other intracellular second messengers also play a part. The generation of platelet Ca2+ signals involves Ca2+ from two sources: stored Ca2' released from the intracellular dense tubular system and extracellular Ca2' entering across the plasma membrane. The mechanisms entailed appear to have much in common with those operating in other non-excitable cells. Platelets are thus a good model for the study of calcium signalling in normal human cells.

Role of nitric oxide in regulation of leucocyte‐endothelial cell interactions

Abstract: Inflammation is a localized response induced by stimuli such as infection and/or cellular and tissue injury. Although inflammation is normally an appropriate response to potentially pathogenic stimuli, under certain conditions, such as reperfusion injury, and in chronic inflammatory processes, such as various forms of arthritis, inflammatory bowel disease and complications associated with sepsis, inappropriate regulation of the inflammatory response can cause abnormal recognition of the host as 'foreign' or absence of a 'termination' signal in an otherwise normal inflammatory process. Critical to the onset of these disease states is the recruitment of leucocytes, which are thought to cause the vascular and tissue injury. For leucocytes to enter tissue they must undergo a cascade of events, mediated by a range of adhesion molecules expressed on the cell membranes of both endothelial cells and leucocytes (Fig. 1). Initially leucocytes leave the mainstream of blood flow and commence rolling at a reduced velocity along the endothelial surface. This process, termed leucocyte rolling, is mediated by three members of the selectin family of adhesion molecules, namely P-selectin, E-selectin and L-selectin. P-selectin, expressed on endothelial cells and platelets, is mobilized to the endothelial cell surface from its storage site in Weibel-Palade bodies within minutes in response to stimuli such as thrombin, histamine and oxidants. Recent evidence from in vivo studies suggests that histamine-induced rolling is entirely dependent on P-selectin (Asako, Kurose, Wolf, DeFrees, Zheng, Phillips, Paulson & Granger, 1994; Kubes & Kanwar, 1994). In addition, cytokines, such as tumour necrosis factor-a (TNF-a) and interleukin-1 (IL-1) can also induce P-selectin expression for periods as long as 24 h, suggesting a role for P-selectin in mediating rolling in more chronic forms of inflammation (Sanders, Wilson, Ballantyne & Beaudet, 1992). E-selectin, also expressed by endothelial cells, has been characterized in vitro to be induced by lipopolysaccharide (LPS), TNF-a and IL-1 (Bevilacqua, Pober, Mendrick, Cotran & Gimbrone, 1987). In response to LPS in vivo, the highest levels of expression are achieved between 3 and 5 h after stimulation (Eppihimer, Wolitzky, Anderson, Labow & Granger, 1996). Despite its demonstrated expression in various inflammatory models, results of studies using either inhibitory antibodies or deletion of the gene responsible for E-selectin expression suggest that E-selectin plays a minimal role in various inflammatory models

The effects of buspirone on perceived exertion and time to fatigue in man

Abstract: Male subjects exercised at 80% maximal rate of O2 uptake (VO2,max) following oral administration of either placebo or the partial 5-HT1A agonist buspirone (45 mg), using a paired design. Ratings of perceived exertion were higher following buspirone and time to volitional fatigue (median and inter-quartile range) fell significantly by approximately a third from 26 min (24-30 min) on placebo to 16 min (11-19 min) following buspirone. Serum prolactin was significantly elevated following buspirone administration, indicating increased hypothalamic 5-HT1A receptor stimulation. There were no significant differences in blood lactate or serum glucose between the trials. This study supports the possible central modulation of exercise tolerance by serotonergic pathways, although a role for dopamine cannot be excluded.

Task-dependent effect of limb immobilization on the fatigability of the elbow flexor muscles in humans.

Abstract: Because short-term limb immobilization produces selective adaptations in the neuromuscular system that probably interact with the task-dependent expression of muscle fatigue, the purpose of this study was to determine the effects of limb immobilization on the ability of human subjects to sustain isometric contractions at low and moderate submaximal forces. Four weeks of elbow joint immobilization caused a substantial decrease in the daily activity of biceps brachii during immobilization, a significant reduction in the cross-sectional area and volume of the elbow flexor muscles as measured by magnetic resonance imaging, and a decline in the maximum voluntary contraction (MVC) activation and force of the elbow flexor muscles. Immobilization had a task-dependent effect on muscle fatigue with a substantially increased endurance time (reduced fatigability) at a low force (20% MVC) and no statistical effect at a moderate force (65% MVC). Despite atrophy of the elbow flexor muscles due to the immobilization, the twitch force elicited in biceps brachii by electrical stimulation was greater after immobilization. The selective improvement of fatigue resistance for the low-force contraction and the absence of a change in the time course of the twitch suggests that the immobilization-induced adaptations included an improved efficacy of some excitation-contraction processes and underscored the major role of these mechanisms in determining the endurance time for low-force, long-duration contractions.

Is human skeletal muscle capillary supply modelled according to fibre size or fibre type

Abstract: Analysis of muscle supply usually relies on estimating either the numerical capillary to fibre ratio or capillary density. Both indices are scale dependent, i.e. they vary with fibre size. We have examined the use of an alternative approach based on the anatomical supply area of individual capillaries, which allows the calculation of a local capillary to fibre ration or density based on area, rather than number of fibres. The results suggest that, in human skeletal muscle, capillary supply is primarily scaled according to fibre size, and is relatively independent of fibre type.

Disruption of vascular signalling by the reaction of nitric oxide with superoxide: implications for cardiovascular disease

Abstract: Oxidation reactions are used in controlled enzymatic pathways to form intracellular messengers such as the prostaglandins, leukotrienes, and the simplest signalling molecule known, nitric oxide. In inflammation, non-enzymatic oxidation appears to produce compounds which compete with or inhibit the mediators derived from endogenous enzymatic oxidative processes. Many of these compounds could be derived from the reaction of NO with superoxide to form peroxynitrite and the ensuing oxidation, nitration and nitrosation reactions. In this short over-view we will describe some of these products and how they may modulate signal transduction events in the vasculature and so contribute to the pathogenesis of atherosclerosis.

Dissociation between metabolic and contractile responses during intermittent isometric exercise in man

Abstract: This study examines the temporal changes in high-energy phosphate and metabolic levels, and in force-generating capacity, during and after voluntary submaximal repetitive isometric exercise (RIE). Eight male subjects performed one-legged RIE with the knee extensors at 40% maximum voluntary contraction (MVC) target force (duty cycle: 6 s contraction, 4 s rest) in a 48 cm bore whole body 1.5 T superconducting magnet. Phosphocreatine (PCr), inorganic phosphate (P(i)), ATP and pH were measured every 9 s. Force-generating capacity was repeatedly measured using MVC force and electrically stimulated contractions (sequential train of impulses of 1-100 Hz). During RIE, MVC declined gradually by 56 +/- 5% (mean +/- S.E.M.). Electrically stimulated force also declined, with a disproportionally large drop in low-frequency force, seen as a decline from 0.76 +/- 0.02 to 0.33 +/- 0.02 in 20:50 Hz force ratio. The PCr decline during RIE was 65 +/- 9%, in most subjects seen as a rapid initial drop followed by less or no further decline to exhaustion. pH declined in parallel by 0.18 +/- 0.04 units, whilst ATP levels remained unchanged throughout the exercise. PCr, P(i) and pH recovered to near control values within 5 min of exhaustion. Force, however, was not fully restored after 30 min recovery. The results support the hypothesis that fatigue from submaximal RIE is unrelated to changes in P(i) and H+ levels. The decline in 20:50 Hz force ratio implies that fatigue may be associated with excitation-contraction coupling impairment. No sudden changes were observed in mechanical or metabolic factors at exhaustion. Exhaustion was probably not caused by lack of substrates for ATP resynthesis, since pH had decreased only marginally.

Modulation of vascular tone by low density lipoproteins: effects on L‐arginine transport and nitric oxide synthesis

Abstract: Low density lipoprotein (LDL) plays an important role in atherogenesis. Focal accumulation within the arterial intima of excess amounts of cholesterol-rich LDL leads to the migration and recruitment of monocytes, which then differentiate into macrophages after taking up large amounts of oxidatively modified LDL via their scavenger receptors and become lipid-laden 'foam cells' within the subendothelial space. It is generally accepted that oxidized LDL and hyperlipidaemia impair endothelial-dependent vascular relaxation, yet the existing literature on the effects of oxidatively modified LDL on endothelium-derived nitric oxide (NO) and prostacyclin (PGI2) release is inconclusive, since oxidized LDL has been reported to enhance or reduce NO and PGI2 production. Our studies using cultured human endothelial and smooth muscle cells have established that basal rates of L-arginine (NO precursor) transport, NO and PGI2 production and soluble guanylyl cyclase activity are unaffected by pretreatment (for 1 or 24 h) with native LDL, or with mildly or highly oxidized LDL. In contrast, highly oxidized LDL inhibited histamine-stimulated release of NO and PGI2 from human endothelial cells and induced an adaptive increase in the level of intracellular glutathione in human smooth muscle cells, a response which was prevented by the chain-breaking antioxidant alpha-tocopherol. Although initial rates of L-arginine transport and basal NO and PGI2 release from human endothelium are unaffected by oxidized LDL, agonist-stimulated release of these vasodilators is markedly attenuated. Elucidation of the mechanisms regulating these responses and their sensitivity to dietary antioxidants could lead to alternative strategies for reducing atherogenesis.

Relationship between membrane potential, delayed rectifier K+ currents and hypoxia in rat pulmonary arterial myocytes

Abstract: Pulmonary arteries constrict in response to hypoxia, a process thought to involve oxygen sensing by K+ channels. We therefore investigated the effects of hypoxia on voltage-activated K+ currents in myocytes isolated from rat small pulmonary arteries using the patch-clamp recording technique. Experiments with iberiotoxin and intracellularly applied Ca2+ chelating agents revealed that hypoxia (PO2, 20–30 mmHg; throughout) inhibited the Ca(2+)-insensitive component of the delayed voltage-activated outward K+ current. Hypoxia did not affect the membrane potential of these cells until they were depolarized by extracellular application of 20 mM K+, current injection or endothelin-1. Hypoxia caused little depolarization in the presence of prostaglandin F2 alpha, an agonist which was ineffective at inducing depolarization. These results suggest that an initial ‘priming’ depolarization may confer a sensitivity to hypoxia by activating delayed rectifier (Kv) channels. Once active, these channels can then be closed by hypoxia, leading to further depolarization. It is unlikely, therefore, that Kv channels are involved in controlling the resting membrane potential of these cells.

Lipid and lipoprotein modification by advanced glycosylation end-products: role in atherosclerosis.

Abstract: When compared with the general population, individuals with diabetes mellitus suffer a 3to 4-fold increased risk of developing atherosclerosis and vascular insufficiency. Since diabetes afflicts at least twelve million individuals in the USA alone, the contribution of hyperglycaemia and insulin resistance to the overall mortality of heart disease and stroke is considerable (Kannel & McGee, 1979; Ruderman & Haudenschild, 1984). Among the pathological processes which have been identified as being important in the development of atherosclerosis are biochemical modifications that affect the functional integrity of low-density lipoprotein (LDL) (Steinberg, Parthasarathy, Carew, Khoo & Witztum, 1989). A dyslipidaemia characterized by increased levels of LDL, very low-density lipoprotein (VLDL) and intermediate-density lipoprotein (IDL) occurs commonly in diabetes and significantly increases the likelihood that these patients will suffer from the atherosclerotic complications of heart attack and stroke (Jensen, Stender & Deckert, 1988; Brown, 1994). Lipoproteins isolated from diabetic plasma have also been shown to exhibit important qualitative abnormalities, such as an impaired capacity to be taken up by fibroblast or tissue LDL receptors (Lopes-Virella, Sherer, Wohltmann, Lees, Mayfield, Sagel, LeRoy & Colwell, 1982; Hiramatsu, Bierman & Chait, 1985; Brown, 1994). The precise molecular basis for this abnormality in uptake is unknown but is of considerable interest since persistent elevation of either LDL or its metabolic precursor, VLDL, has been shown to be an important risk factor across different populations for the development of atherosclerosis (Goldstein & Brown, 1977; Kannel & McGee, 1979; Ruderman & Haudenschild, 1984; Steinberg et al. 1989).

Pituitary adenylyl cyclase-activating polypeptides and vasoactive intestinal peptide inhibit bone resorption by isolated rabbit osteoclasts

Abstract: Nerve fibres present in the periosteum, cortical bone and bone marrow are proposed to be involved in the regulation of bone metabolism by the release of neuropeptides acting locally on bone cells. The present study describes the effects of vasoactive intestinal polypeptide (VIP), shown to be present in the vicinity of bone, and the two related neuropeptides pituitary adenylyl cyclase-activating polypeptide(1-38) (PACAP-38) and PACAP-27 on bone resorption in vitro induced by osteoclasts isolated from 10-day-old rabbits. Bone resorption was measured as the number and total area of pits formed by tartrate-resistant acid phosphatase-positive multinucleated cells (TRAP + MNCs) cultured for 3 days on devitalized slices of bovine cortical bone. All three neuropeptides had significant inhibitory effects on both the number and area of pits formed. At a high concentration (10(-7) M) the mean +/ࢤ S.E.M. reductions in the total area resorbed compared with controls were 70.3 +/ࢤ 8.2, 45.2 +/ࢤ 7.3 and 63.4 +/ࢤ 7.2% for PACAP-38, PACAP-27 and VIP, respectively. The corresponding values for the apparent dissociation constants were 0.93 +/ࢤ 0.30, 3.2 +/ࢤ 1.6 and 0.35 +/ࢤ 0.14 nM, respectively. The number of TRAP + MNCs was unaffected by application of neuropeptides. Autoradiography showed the presence of 125I-VIP binding sites on some stromal cells, whereas osteoclasts had no binding sites for 125I-VIP. A high number of 125I-calcitonin binding sites was demonstrated on osteoclasts in the same preparation. The signal transduction pathway remains to be fully elucidated but seems to be partly dependent on changes in intracellular calcium concentrations, since a number of stromal cells responded to application of 10(-8) M PACAP-38 or VIP, and at least partly independent of cAMP accumulation. Trifluoperazine and mellitin, two selective calmodulin inhibitors, failed to block the VIP-induced inhibition of bone resorption. Our results demonstrate a non-toxic and probably stromal cell-derived effect of PACAP-38, PACAP-27 and VIP on isolated rabbit osteoclasts, resulting in a potent inhibition of bone resorption in vitro. The signal transduction pathway for inhibition induced by PACAP-38, PACAP-27 and VIP may be mediated partly by changes in intracellular Ca2+ in stromal cells.

Gadolinium blocks the delayed rectifier potassium current in isolated guinea-pig ventricular myocytes

Abstract: The effect of Gd3+ on the delayed rectifier potassium current (IK) in single guinea-pig ventricular myocytes was tested using whole-cell patch-clamp techniques. It was found that Gd3+ blocked 70% of the IK tail current at a concentration of 100 microM. The EC50 was 24 microM. Action potential durations were, however, reduced, consistent with a predominant effect on depolarizing L-type Ca2+ current (Ica.L). In the presence of 5 microM nifedipine Gd3+ prolonged the action potential. Using carbon fibres to stretch cells we observed that 10 microM Gd3+ was not effective in reducing a large stretch-activated increase in resting calcium. Modelling studies using the OXSOFT HEART program suggest that this lack of response is influenced by blockade of repolarizing current but is best reproduced by additional blockade of Ca2+ extrusion via the Na(+)ࢤCa2+ exchanger. When Gd3+ is used as a blocker of stretch-activated channels its actions upon both Ica.L and IK must therefore be accounted for.

Anion fluxes, volume regulation and electrical activity in the mammalian pancreatic beta‐cell

Abstract: The secretion of insulin from the pancreatic f-cell can be influenced, as with numerous endocrine cell types, by hormones, neurotransmitters and drugs which bind to cell surface receptors and generate an intracellular signal (e.g. cyclic AMP, inositol 1,4,5-trisphosphate). However, the ,-cell has in addition the uncommon, possibly unique, ability to increase its hormone output in response to a rise in the ambient concentration of nutrients, notably glucose. Insulin release in response to virtually any type of stimulus is intimately associated with altered electrical activity. For example, a rise in glucose concentration within the approximate range 5-20 mM results in a progressive increase in the intensity of electrical, and hence secretory, activity. This, and virtually all other effects of glucose on the fl-cell, require metabolism of the sugar and the generation of some intracellular metabolic signal(s), the precise identity of which is unknown. The nature and regulation of/-cell electrical activity is a complex subject which has been extensively reviewed (for example, see Ashcroft & Rorsman, 1989; Cook, Satin & Hopkins, 1991). The majority of studies of f-cell electrophysiology have employed mouse islets or f-cells, and it is becoming apparent that profound species differences exist. In mouse fl-cells exposed to intermediate glucose concentrations, a rhythmic 'bursting' pattern is seen, consisting of a depolarization of the membrane potential, leading to bursts of Ca2+-dependent action potentials followed by a repolarization leading to a silent phase. As the glucose concentration progressively rises, the duration of the bursts

Identification of projections from the central nucleus of the amygdala to the paraventricular nucleus of the hypothalamus which are immunoreactive for corticotrophin-releasing hormone in the rat

Abstract: Corticotrophin-releasing hormone (CRH) immunoreactivity was detected in neurons of the central nucleus of the amygdala that were retrogradely labelled by injection of a fluorescent tracer (True Blue) into the paraventricular nucleus of the hypothalamus (PVN). The double-labelled neurons were located mainly in the medial subdivision of the central nucleus and appeared to comprise less than one-fifth of the descending pathway. These results suggest that CRH may act as a neurotransmitter in the amygdalo-hypothalamic pathway.

Responses of neurones in the medullary raphe nuclei to inputs from visceral nociceptors and the ventrolateral periaqueductal grey in the rat

Abstract: The ventrolateral periaqueductal grey matter (PAG) is believed to have a role in mediating cardiovascular responses to noxious visceral stimuli. The present study was carried out as a first stage in establishing whether the ventrolateral PAG may exert these influences after a relay in the caudal medullary raphe nuclei (nucleus raphe obscurus and nucleus raphe pallidus). Single unit extracellular recordings were made from neurones in the caudal raphe nuclei and, for comparison, in the more rostral nucleus raphe magnus in Saffan-anaesthetized and paralysed rats. Neurones in the mid-line medulla were tested for their responses to electrical stimulation at chemically identified depressor sites in the ventrolateral PAG and to noxious visceral stimuli (distensions of the urinary bladder and electrical stimulation of the greater splanchnic nerve). Fifty-two per cent of caudal and 74% of rostral mid-line neurones gave short latency excitatory responses to stimulation of depressor sites in the ventrolateral PAG. Of the neurones that were also tested with noxious visceral stimuli, 5% of the caudal and 47% of the rostral neurones responded to bladder distension, while 33 and 35%, respectively, of caudal and rostral neurones responded to splanchnic nerve stimulation. These results indicate that many mid-line medullary neurones receive inputs from both the ventrolateral PAG and visceral nociceptors and may, therefore, be part of the output pathway by which the ventrolateral PAG produces integrated physiological responses to noxious visceral stimuli.

Nitric oxide in the control of submandibular gland function in the anaesthetized ferret

Abstract: Stimulation of parasympathetic innervation of the submandibular gland (2 or 20 Hz continuously or 20 Hz for 1 s at 10 s intervals), in the ferret, produced secretion of fluid and protein and a fall in vascular resistance. The responses following the administration of N omega-nitro-L-arginine methyl ester (L-NAME; 2 mmol kg-1 i.a.) to block the synthesis of nitric oxide (NO) were reduced, and the persisting responses were abolished (at 2 Hz continuously and 20 Hz intermittently) or further reduced (at 20 Hz continuously) by the additional administration of atropine. The output of vasoactive intestinal peptide (VIP) from the gland was not affected. Neither the secretory nor the vascular response to intra-arterial infusions of acetylcholine (1.25 nmol kg-1) was affected by L-NAME, whereas the vascular responses to both VIP (10 pmol kg-1) and pituitary adenylate cyclase-activating peptide (1-38) (PACAP) (0.5 pmol kg-1) were reduced thereby. Neither peptide evoked a fluid secretion per se. However, when infused during parasympathetic stimulation of saliva secretion, VIP increased both flow rate and the output of protein. These effects of VIP were abolished by L-NAME. The experiments were performed in the presence of sodium nitroprusside at doses (4-8 nmol min-1 kg-1 i.v.) aimed to counterbalance the systemic effects of L-NAME. The results show that, in the ferret, parasympathetic nerve activity increases submandibular blood flow, and elicits the flow of saliva and output of protein by mechanisms that involve in situ generation of NO, upon which the effects of VIP and PACAP but not acetylcholine are largely dependent.

Glycosaminoglycan depletion greatly raises the hydraulic permeability of rabbit joint synovial lining

Abstract: The hydraulic resistance of the synovial lining of a joint is important for retention of intraarticular lubricant. The resistance has been attributed to synovial interstitial glycosaminoglycans. This was tested by depletion of hyaluronan and chondroitin sulphates from synovium in five rabbit knees in vivo under anaesthesia, using testicular hyaluronidase. The enzyme raised synovial permeability to fluid 5- to 7-fold-substantially more, in fact, than predicted by a recent model. The results prove that hyaluronan and/or chondroitin sulphate are important sources of hydraulic resistance in synovium.

An early transient current is associated with hyposmotic swelling and volume regulation in embryonic chick cardiac myocytes

Abstract: Hyposmotically induced changes in membrane conductance were measured in embryonic chick cardiac myocytes using conventional and perforated patch-clamp recording techniques; simultaneous measurements of cell volume were made from the video image of the voltage-clamped cell. Hyposmotic challenge was associated with a rapid, transient current coincident with the onset of cell swelling; cell volume subsequently recovered towards control values (regulatory volume decrease; RVD). The transient swelling-induced current (I(swell)) reversed at +15 mV, and was not found to be carried exclusively by any single ion in the physiological solutions. I(swell) was abolished by gadolinium (Gd3+), a blocker of stretch-activated ion channels, and was absent when the cytoskeleton was disrupted by treatment with cytochalasin B. I(swell) was also prevented when intracellular [Ca2+] was buffered with BAPTA AM. Under those experimental conditions which prevented the generation of I(swell), cell volume regulation failed so that the cells remained swollen in hyposmotic solution. Our data reveal a functional relationship between I(swell) and RVD, whereby I(swell) is a necessary prerequisite, although not exclusively sufficient, for volume recovery following cell swelling. We propose that I(swell) is an important early signalling event which activates subsequent mechanisms to regulate cell volume.