bluuebunny/crossref_metadata_2025 huggingface dataset:
Osteoclast demise in the rat: physiological versus degenerative cell death
Authors: Lutton JD , Moonga BS , Dempster DW
Published: December 2014

Time‐lapse video microscopy was used to assess the temporal morphological events undergone by rat osteoclasts after exposure to a variety of agents that promote cell death. Direct observations revealed that there were two morphologically distinguishable forms of cell death, which resembled apoptosis and necrosis, respectively. Marked changes in morphology became apparent after 2–4 h exposure to a variety of agents, including cyclosporine A, tamoxifen, corticosterone and dexamethasone. The cells began to shrink rapidly and within 25 min appeared as small round spheres. At this time, the cell membranes underwent violent distortive boiling, or zeiosis, accompanied by the formation of small membrane buds. The cells maintained a spherical configuration and the membrane appeared to remain intact for several hours. These observations are consistent with the process of physiological cell death or apoptosis. In some cases, post‐apoptotic changes or secondary necrosis could be seen, including membrane blebbing and degeneration. In contrast, when cells were exposed to hydrogen peroxide or sodium azide, there was a marked deterioration of the cell membrane after 1–4h. This included the formation of spikes and/or blebbing with the release of intracellular debris, resulting in an overall spattered appearance. This type of appearance is characteristic of degenerative cell death or necrosis. The significance of the mode of osteoclast death is discussed.
Depletion of vasoactive intestinal peptide and substance P in parotid glands of atropinized rats during reflex secretion
Authors: Asztely A , Ekman R , Tobin G , Ekstrom J
Published: December 2014

Rats previously starved for 30 h were offered hard chow over a period of 80 min, and the effect of eating on the parotid gland content of vasoactive intestinal peptide (VIP), substance P and neuropeptide Y (NPY) was examined. In rats pretreated with I.P. atropine and alpha‐ and beta‐adrenoceptor blocking agents, the total amounts of VIP and substance P were reduced after feeding by 23 and 42%, respectively; in contrast the NPY content was not significantly affected. Similarly, in animals given atropine alone the VIP content was reduced by 25% and substance P by 40% after feeding. In the absence of autonomic receptor blockade, there was no loss of parotid neuropeptide content in response to feeding. Neither an intact sympathetic innervation nor a capsaicin‐sensitive sensory innervation was a prerequisite for depletion of parotid neuropeptides in the presence of atropine and adrenoceptor blockers. The observed decrease in neuropeptide content is thought to reflect an imbalance between peptide release from nerve terminals and their replenishment by axonal transport, this imbalance resulting from the unusually high demands on the parasympathetic non‐adrenergic, non‐cholinergic (NANC) mechanism. Under normal conditions salivary peptidergic mechanisms are spared as seen in the absence of autonomic receptor blockade.
The effects of unilateral and bilateral chewing, empty clenching and simulated bruxism, on the masticatory‐parotid salivary reflex in man
Authors: Anderson DJ , Hector MP , Linden RW
Published: December 2014

In man, parotid flow has been recorded, using modified Lashley cups, in response to mechanical stimulation of the teeth by repeated chewing and clenching manoeuvres. The rectified and integrated masseter electromyographic activity was used as an indirect measure of the stimulus intensity where necessary. Following the observation that –empty clenching— failed to evoke a parotid salivary response, a series of three experiments has been undertaken to address the following questions. (1) Is a lateral component of force required to evoke a flow during empty clenching? (2) Does contralateral inhibition of salivary secretion explain this observation? (3) What is the threshold for the masticatory‐salivary reflex? The results suggest that: (i) empty clenching together with a lateral component of force does not result in a secretion above resting flows; (ii) there is no evidence for the existence of contralateral inhibition of salivary secretion; and (iii) the threshold for the masticatory‐salivary reflex is lower than 5% of comfortable chewing forces. We are still unable to offer an explanation for the lack of parotid secretion during empty clenching.
Non‐quantal acetylcholine release in the mouse diaphragm after phrenic nerve crush and during recovery
Authors: Nikolsky EE , Oranska TI , Vyskocil F
Published: December 2014

The progressive decline and recovery of spontaneous quantal acetylcholine (ACh) release (miniature endplate potentials, MEPPs) and the H‐effect were measured in the mouse diaphragm after nerve crush and during regeneration. The H‐effect is the hyperpolarization of the muscle fibre membrane produced by the addition of (+)tubocurarine, which indicates non‐quantal ACh release. One hour after nerve crush the H‐effect had declined to 50% of control values and 4 h later the H‐effect disappeared completely. There were no substantial changes in the MEPP frequency and amplitude during the first 4 h after denervation. MEPP frequency then increased, but after 6 h of denervation it decreased and after 16 h no MEPPs were found in any of the muscle fibres. The times of onset of these denervation changes in the proximal, central and distal parts of diaphragm were similar. During reinnervation, the H‐effect was detectable in all muscle parts 3 days before the reappearance of MEPPs. The H‐effect developed first on day 8 in the proximal endplates and then, with a delay of 3 and 6 days, in the central and distal areas, respectively. During axonal regrowth the non‐quantal release was restored before detectable quantal release. Non‐quantal release is the first indication of the ability of the nerve terminal to release ACh in the process of reinnervation.
Characterization of a sodium‐dependent magnesium efflux from magnesium‐loaded rat pancreatic acinar cells
Authors: Wisdom DM , Geada MM , Singh J
Published: December 2014

This study investigates the mechanism of magnesium (Mg2+) transport (efflux) from the exocrine rat pancreas. Permeabilized pancreatic acini were loaded with Mg2+ by employing a high‐Mg2+ (12 mM) buffer containing A23187 (6 microM). Net Mg2+ efflux was measured using the technique of atomic absorbance spectrophotometry. Incubation of preloaded acini in a buffer deficient in Mg2+ resulted in a large and time‐dependent release of Mg2+ with maximal efflux occurring within 40 min. Pretreatment of loaded acini with bumetanide, SITS or ouabain had no significant effect on Mg2+ efflux. In contrast, when acini were pretreated with 10 mM dinitrophenol, 10(‐4) M amiloride, 1 mM lidocaine or 1 mM quinidine there were significant (P < 0.001) decreases in net Mg2+ efflux. Replacement of extracellular sodium [Na+]o with either N‐methyl‐D‐glucamine (NMDG), Tris or choline resulted in a significant (P < 0.001) inhibition of Mg2+ efflux. The results of this study indicate that Mg2+ transport (efflux) in pancreatic acinar cells may not be associated with the Na(+)‐K(+)‐ATPase, the Na(+)‐K(+)‐Cl‐ cotransporter or the anion exchanger, but with a Na(2+)‐sensitive Mg2+ transport system.
Modulation of vagal actions on heart rate produced by inhibition of nitric oxide synthase in the anaesthetized ferret
Authors: Conlon K , Collins T , Kidd C
Published: December 2014

We have examined a possible role for nitric oxide in the efferent vagal control of heart rate of the anaesthetized ferret, using stimulation of the motor fibres of the right vagus at supramaximal intensities and increasing frequencies. Propranolol was used to block beta‐adrenergic actions on the heart. L‐NAME (50–100 mg i.v.) significantly reduced the bradycardia induced by vagal stimulation at all frequencies tested (1–15 Hz); subsequent infusion of L‐arginine (100–200 mg i.v.) restored the vagally induced heart rate responses to control levels. These results strongly suggest a role for nitric oxide in modulating the vagally mediated control of heart rate.
On the role of bicarbonate as a hydrogen ion buffer in rat CNS neurones
Authors: Amos BJ , Pocock G , Richards CD
Published: December 2014

The role of bicarbonate as a hydrogen ion buffer has been investigated using the fluorescent dye BCECF in individual rat cerebellar, hippocampal and neocortical neurones maintained in culture. The steady‐state intracellular pH (pHi) was estimated to be 7.07 +/− 0.05 (n = 22) in CO2‐HCO3(‐)‐buffered media. Buffering power (beta) estimated from the addition and removal of weak bases was ca 10 mM (pH unit)‐1 and was found to be similar in both CO2‐HCO3(‐)‐ and Hepes‐buffered media. The membrane‐permeant carbonic anhydrase inhibitor, acetazolamide (10–20 microM), did not affect estimates of beta. The results indicate that CO2‐HCO3‐ does not act as an open buffer system in these neurones.
Effect of ileal infusion of volatile fatty acids on feed intake and urinary magnesium excretion in sheep
Authors: Dalley DE , Sykes AR , Isherwood P , Robson AB
Published: December 2014

Five 2‐year‐old female sheep fitted with cannulae in the terminal ileum and proximal colon were infused, intra‐ilealy during a 24 h period, with volatile fatty acids (VFA) (ratio of acetic:propionic:butyric acid, 0.80:0.15:0.05) at rate of 0, 220, 440, 660 or 880 mmol/day in 21 deionized water. Daily feed intake was progressively depressed by VFA and reached 87 +/− 4.7% (S.E.M.) at the highest rate of infusion. Colonic digesta pH decreased by 2.1 +/− 0.48 units and Mg solubility increased from 21 +/− 2.6 to 52 +/− 1.2% 16 h after the start at the highest rate of VFA infusion. Urinary Mg excretion increased during the first 4 h of infusion on all treatments but the increase was not sustained. The results are discussed in the context of limited knowledge of the importance of the large intestine in intake regulation and Mg absorption.
Ion channels in freshly isolated and cultured human bronchial smooth muscle cells
Authors: Snetkov VA , Hirst SJ , Ward JP
Published: December 2014

Voltage‐gated ion currents were studied in human bronchial airway smooth muscle (ASM) cells. Proliferating or growth‐arrested cells in culture were compared with freshly isolated cells. Three types of charybdotoxin (ChTX)‐sensitive K+ channel were observed in all cell types, with conductances in symmetrical 140 mM KCl solutions ([Ca2+]i < 0.1 nM) of 206 +/‐ 14 pS (n = 32), 144 +/‐ 11 pS (n = 27) and 109 +/‐ 5 pS (n = 25). The relative proportion of each channel type differed substantially between the three groups of cells. In freshly isolated ASM cells large conductance K+ channels were represented almost entirely by a conductance of 206 pS, which was found in all twenty‐three patches studied. In contrast, in most patches from proliferating cells the majority of channels had conductances of either 144 pS (14 of 21 patches) or 109 pS (8 of 21 patches). Cultured cells that had been growth arrested by serum depletion revealed the same set of channels as the proliferating cells, but the occurrence of the 109 pS channel was much more frequent (16 of 19 patches). As has been shown previously, 206 pS channels were active at a physiological membrane potential (‐60 to ‐20 mV) even at a very low free [Ca2+]. The 144 pS channels could be recorded only at depolarized potentials (+80 to +100 mV), whereas 109 pS channels were active over a wide range of potentials (‐60 to +100 mV), but only in the presence of GTP. In a proportion of cultured cells a tetrodotoxin‐sensitive Na+ current and a hyperpolarization‐induced inwardly rectifying K+ current were also observed (15 and 21%, respectively, of all cells examined). Neither of these currents were observed in freshly isolated cells. Whole‐cell outward current in all groups was sensitive to tetraethylammonium, ChTX, and iberiotoxin, but not to 4‐aminopyridine. In summary, it is clear that during proliferation there are considerable changes in the expression of ionic channels in ASM that have profound functional significance. In particular, these changes would tend to make the tissue more excitable, and may be of relevance to the proliferative process itself.
Frequency and degree of milk removal and the short‐term control of human milk synthesis
Authors: Daly SE , Kent JC , Owens RA , Hartmann PE
Published: December 2014

The effect of the temporal spacing and degree of milk removal by either expression or breastfeed on short‐term (hour to hour) rates of milk synthesis was investigated. For four expressing mothers (three of whom had given birth prematurely), the relationship between milk produced at an expression and the time since the last expression showed that proportionally less milk is produced after intervals of 6‐18 h than after intervals of less than 6 h (P < 0.0002). For five breastfeeding and four expressing mothers (three of whom had given birth prematurely), rates of milk synthesis (ranging from 0 to 56 ml/h) were linear between breastfeeds and expressions (intervals ranging from < 1 h to 6 h).Changes in rate of milk synthesis were not directly associated with the frequency of breastfeeds but rather with the degree of emptying of the breast (P < 0.05). These results indicate the importance of degree, rather than frequency, of breast emptying in the short‐term control of human milk synthesis.
Truncated glucagon‐like peptide−1 and oxyntomodulin stimulate somatostatin release from rabbit fundic D‐cells in primary culture
Authors: Beales IL , Calam J
Published: December 2014

The ability of the proglucagon‐derived peptides oxyntomodulin and truncated glucagon‐like peptide‐1 (7−36)amide to stimulate somatostatin release from cultured rabbit fundic D‐cells was assessed. Significant stimulation of somatostatin release was seen with both peptides at 10 nM. Potentiation of the stimulatory action of cholecystokinin was seen with both peptides at both 100 pM and 10 nM. The paracrine release of somatostatin from fundic D‐cells may explain the acid inhibitory actions of the enteroglucagons. The different small bowel‐derived peptides may interact at the level of the D‐cell to regulate gastric acid secretion.
The occurrence of stable subconductance levels in Na(+)‐activated K+ channels in excised membrane patches from guinea‐pig ventricular myocytes
Authors: Mistry DK , Tripathi O , Chapman RA
Published: December 2014

Twelve short‐lived subconductance levels have been recognized in potassium channels activated by intracellular Na+ (KNa channels) in membrane patches excised from guinea‐pig ventricular myocytes. When stable recording was achieved over a prolonged period (> 30 min), sojourns of opening to one of two stable subconductance levels (approximately 30 and 70% of the main‐state level) occurred spontaneously. These periods of activity showed similar kinetics (open and closed time distributions) to those of openings to the main‐state conductance. Opening to transient sublevels was still detected during openings of the channel to the stable sublevels. These results suggest that the KNa channel has a complex oligomeric structure, in which twelve separate pores would seem to be functionally organized into either three or four domains that show a high degree of co‐operativity in their gating.
Effect on phloridzin on net rate of liquid absorption from the pleural space of rabbits
Authors: Zocchi L , Agostoni E , Raffaini A
Published: December 2014

Previous indirect findings have suggested the occurrence of solute‐coupled liquid absorption from the pleural space, consistent with Na(+)‐K(+)‐ATPase on the interstitial side plus a Na(+)‐H+ and CI(‐)‐HCO3‐ double exchange on the luminal side of the pleural mesothelium. To assess whether Na(+)‐glucose cotransport also operates on the luminal side, the relationship between net rate of liquid absorption from the right pleural space (Jnet) and volume of liquid injected into this space (0.5, 1 or 2 ml) was determined in anaesthetized rabbits during hydrothoraces with phloridzin (10(‐3)M) or with phloridzin plus 4‐acetamido‐4'‐isothiocyanatostilbene‐2, 2'‐disulphonic acid (SITS; 1.5 x 10(‐4)M). The relationship obtained during hydrothoraces with phloridzin was displaced downwards by 0.09 ml h‐1 relative to that in control hydrothoraces (P < 0.01). The decrease in Jnet was similar in hydrothoraces of various sizes. The relationship obtained in hydrothoraces with phloridzin plus SITS was displaced downwards by 0.16 ml h‐1 relative to that in control hydrothoraces (P < 0.01), i.e. the decrease in Jnet was similar to the sum (0.17 ml h‐1) of the decreases in Jnet produced individually by phloridzin and by SITS (0.08 ml h‐1). The decrease in Jnet was similar in hydrothoraces of differing size. The above findings are consistent with the occurrence of Na(+)‐glucose cotransport on the luminal side of the pleural mesothelium, operating simultaneously with the double exchange also under physiological conditions.
Effect of rearing temperature on perirenal adipose tissue development and thermoregulation following methimazole treatment of postnatal lambs
Authors: Symonds ME , Andrews DC , Buss DS , Clarke L , Darby CJ , Lomax MA
Published: December 2014

This study examined the effect of ambient temperature on perirenal adipose tissue development and thermoregulation over the first month of postnatal life in lambs treated with a drug that reduces thyroid hormone synthesis (methimazole; at a dose of 50 mg day‐1 (kg body weight)‐1). Twin lambs were hand‐reared at a fixed level of nutrition in either a warm (WR; 25 degrees C) or cool (CR; 10‐15 degrees C) ambient temperature. Oxygen consumption, heart rate and colonic temperature were measured during non‐rapid eye movement sleep in different ambient temperatures (6, 15, 25 and 34–36 degrees C) at 7, 14 and 28 days of age. Plasma thyroid hormone concentrations decreased with postnatal age and were higher in CR than in WR lambs. All lambs increased plasma thyroid hormone concentrations and O2 consumption during cold exposure but this was associated with a mean increase in colonic temperature in WR lambs at 7 or 28 days. Colonic temperature increased with the onset of panting at all ages when lambs were exposed to 34–36 degrees C, a response that was greatest at 28 days in CR lambs. An increase in colonic temperature with age was observed in CR but not WR lambs. Heart rate declined with age only in the WR group. At 8 and 29 days there were no significant differences between WR and CR groups in the occurrence of shivering or in the thermogenic activity (i.e. GDP binding to mitochondria) of perirenal adipose tissue. At 29 days WR lambs possessed more adipose tissue with a higher lipid and DNA content. It is concluded that modest changes in rearing temperature can have a large influence on the control of body temperature and thyroid hormone response to methimazole treatment. CR lambs are able to maintain higher plasma thyroid hormone concentrations and exhibit improved thermoregulation compared with WR lambs without any detectable differences in brown adipose tissue function.
Organization of the sural cutaneous input regulating the discharge of triceps surae gamma‐motoneurones in the cat
Authors: Ellaway PH , Davey NJ , Ljubisavljevic M
Published: December 2014

The organization of the cutaneous afferent influence on the discharge of gamma‐motoneurones has been investigated in the decerebrated, spinal cat. gamma‐Motoneurone discharges were recorded from cut nerve filaments. Time and frequency domain analyses were used to reveal the strength of coupling between gamma‐motoneurone discharge and cutaneous afferents excited by natural skin stimulation. Time domain analysis (cross‐correlation) was also used to reveal the sigh (facilitation or inhibition) and time course of the cutaneous influence on individual gamma‐motoneurones. Mechanical stimulation of discrete areas of skin within the sural nerve field caused facilitation or inhibition of individual gamma‐motoneurones supplying the gastrocnemius and soleus muscles. In a few cases, a gamma‐motoneurone facilitated by stimulation at one site could be inhibited from another location. The effect of cutaneous afferent stimulation was not evident in the decerebrated cat with intact spinal cord. The intensity of facilitation and inhibition was mapped for the sural nerve field. Facilitation had focus of highest intensity to stimulation applied between the calcaneum and lateral malleolus. The focus for inhibition was either the same as for facilitation or, more frequently, tended to be lateral and dorsal to the calcaneum at the edge of the sural field. Cutaneous stimulation at the edge of the sural field could also reduce the coherence between the discharges of gamma‐motoneurones, particularly at low frequencies of association (1–5 Hz), indicating disfacilitation of other sources of afferent input. The results reveal a detailed pattern of cutaneous inputs to the fusimotor system that could participate in a wide range of behavioural adjustments to stretch or contact of the skin at the heel.
Dissociation between metabolic and contractile responses during intermittent isometric exercise in man
Authors: Saugen E , Vollestad NK , Gibson H , Martin PA , Edwards RH
Published: December 2014

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.
Is human skeletal muscle capillary supply modelled according to fibre size or fibre type?
Authors: Ahmed SK , Egginton S , Jakeman PM , Mannion AF , Ross HF
Published: December 2014

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.
L‐type calcium current in catecholamine‐induced cardiac hypertrophy in the rat
Authors: Meszaros J , Coutinho JJ , Bryant SM , Ryder KO , Hart G
Published: December 2014

This study investigates whether an increase in L‐type calcium current (ICa) could explain the prolongation of the action potential associated with the cardiac hypertrophy produced by repeated administration of isoprenaline. Hypertrophy was induced by daily injection of isoprenaline (5 mg/kg i.p.) for 7 days in male Wistar rats. Under whole‐cell voltage‐clamp conditions, ICa was evoked in Na(+)‐ and K(+)‐free solution, by step depolarizations from a holding potential of ‐45 mV in single left ventricular myocytes isolated from control and hypertrophied rat hearts. In the test group, heart weight to body weight ratio and cell membrane capacitance were increased by 30 and 34%, respectively. Peak ICa was increased by 26% (control, ‐1.46 +/− 0.06 nA, n = 17; hypertrophy, ‐1.85 +/− 0.13 nA, n = 19; P < 0.02). However, when normalized for cell capacitance, there was no significant difference in peak current density (control, ‐12.1 +/− 0.5 pA/pF; hypertrophy, ‐11.5 +/− 0.6 pA/pF). The voltage dependence of ICa was similar in both cell types. No change was observed either in the steady‐state activation or inactivation kinetics, or in the time course of inactivation. The recovery from inactivation of ICa, when fitted with monoexponential function with time constant tau rec, was not changed significantly by hypertrophy (control, tau rec = 115 +/− 23 ms, n = 9; hypertrophy, tau rec = 120 +/− 12 ms, n = 15). The increased calcium current occurs in parallel with the increase in cell size. The prolonged action potential duration seen in this model must be explained by changes in currents other than L‐type calcium current.
Disruption of vascular signalling by the reaction of nitric oxide with superoxide: implications for cardiovascular disease
Authors: Darley‐Usmar V , White R
Published: December 2014

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.
Modulation of vascular tone by low density lipoproteins: effects on L‐arginine transport and nitric oxide synthesis
Authors: Jay MT , Chirico S , Siow RC , Bruckdorfer KR , Jacobs M , Leake DS , Pearson JD , Mann GE
Published: December 2014

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.
Placental transporter activity and expression in relation to fetal growth
Authors: Sibley C , Glazier J , D'Souza S
Published: December 2014

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.
Engine and radiator: fetal and placental interactions for heat dissipation
Authors: Schroder HJ , Power GG
Published: December 2014

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, 1.1 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 0.5 degree C (0.3‐1 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, e.g. 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.
Calibration of Mg(2+)ࢤselective macroelectrodes down to 1 mumol lߝ1 in intracellular and Ca(2+)ࢤcontaining extracellular solutions
Authors: Luthi D , Spichiger U , Forster I , McGuigan JA
Published: December 2014

Using Mg(2+)ࢤselective macroelectrodes based on the neutral carriers ETH 7025 and ETH 5506, methods were developed to determine accurately the apparent binding constant (Kapp) and purity, and hence the ionized magnesium concentration ([Mg2+]), in Ca(2+)ࢤfree, Mg2+ buffer solutions manufactured with either CDTA (trans‐1,2‐diaminocyclohexane‐N,N,N',N'‐tetraacetic acid monohydrate) or EDTA. In nominally Ca(2+)‐free solutions, calibration of the macroelectrodes was possible down to 1 mumol l‐1 in both intracellular (ETH 7025)‐ and extracellular (ETH 5506)‐like physiological solutions. The measured [Mg2+] in the buffer solutions overlapped with the [Mg2+] set by dilution alone, suggesting that the method was reliable. These buffer solutions could then be used to manufacture standard Mg2+ solution containing known [Mg2+] at a set calcium concentration. Ca2+ sensitivity limits the use of ETH 7025 and, for extracellular measurements in Ca(2+)ࢤcontaining solutions, Mg(2+)ࢤselective macroelectrodes manufactured with ETH 5506 were the electrodes of choice. In 0.5–0.9 mmol l–1 Ca+, measurement of [Mg2+] was possible down to 10 mumol l‐1. At [Mg2+] greater than 0.25 mmol l‐1 there was no interference from Ca2+ (0.5–1.5 mmol l–1). With CDTA and/or EDTA buffer solutions there was a wide variation between the calculated values for the [Mg2+] and the measured [Mg2+] (the calculated values differed by a factor of up to 4.5 and 3, respectively). At present, measurement of the Kapp and ligand purity in the appropriate solution at the desired pH and temperature would seem to be the best strategy to adopt rather than attempting to calculate the constant. Since no recognized international standard exists for [Mg2+] at the micromolar level, values in the literature for Kd, etc. in this range can only be regarded as approximate.
The effect of cromakalim on intracellular [Ca2+] in isolated rat skeletal muscle during fatigue and metabolic blockade
Authors: Burton FL , Smith GL
Published: December 2014

The effects of the ATP‐sensitive K+ channel (KATP channel) opener cromakalim on excitation‐contraction (E‐C) coupling were studied in skeletal muscle during fatiguing and non‐fatiguing activity. Intracellular calcium concentration ([Ca2+]i) was monitored using the fluorescent indicator fura‐2 in isolated single skeletal muscle fibres enzymatically dissociated from rat flexor digitorum brevis. A protocol of tetanic stimulation (50 Hz for 300 ms) with progressively shorter durations between tetani was used to induce E‐C coupling failure in these cells. Cromakalim (100‐800 microM) had little effect on peak [Ca2+]i during twitch and non‐fatiguing tetanic stimulation. However, with 0.4 s between tetani, 100 microM cromakalim decreased peak tetanic [Ca2+]i from 1.47 +/ࢤ 0.11 microM to 8.35 +/ࢤ 55 nM, but did not affect resting [Ca2+]i (control, 220 +/ࢤ 40 nM; with cromakalim, 171 +/ࢤ 33 nM). Cyanide (2 mM) decreased tetanic [Ca2+]i and increased resting [Ca2+]i during the stimulus protocol; with 0.4 s between tetani, peak [Ca2+]i was 820 +/ࢤ 50 nM and resting [Ca2+]i was 443 +/ࢤ 32 nM. The ability of cromakalim to inhibit E‐C coupling was enhanced by the presence of cyanide. Complete blockade of metabolism by cyanide and iodoactetate (0.1 mM) caused a marked rise in resting [Ca2+]i and inhibition of the tetanic rise of [Ca2+]i. With cromakalim (100 microM) present, E‐C coupling failed during metabolic blockade but without a significant increase in resting [Ca2+]i. These results are consistent with a role for the KATP channel in the failure of Ca2+ release during fatigue.
Anaerobic energy production in human skeletal muscle in intense contraction: a comparison of 31P magnetic resonance spectroscopy and biochemical techniques
Authors: Constantin-Teodosiu D , Greenhaff PL , McIntyre DB , Round JM , Jones DA
Published: December 2014

Five subjects underwent twenty electrically evoked maximal isometric contractions of the anterior tibialis muscle of both legs (n = 10), with limb blood flow occluded. Measurements of muscle high‐energy phosphates (ATP, ADP and phosphocreatine (PCr)), lactate and pH were made using both 31P magnetic resonance spectroscopy (MRS) and the biochemical analysis of biopsy samples obtained from directly below the MRS surface coil. The resting PCr concentration determined using MRS was similar to that measured in the biopsy material. Following contraction, MRS showed a greater decrease in ATP concentration compared with biochemical analysis (P < 0.05), but the decrease in PCr was similar. Good agreement was found when comparing resting muscle pH estimated by the two methods. Post‐exercise muscle pH was, however, consistently lower with MRS and consequently the accumulation of muscle lactate estimated using MRS was markedly greater than the corresponding biochemical measurement (P < 0.05). As a result, MRS revealed an approximately 30% greater anaerobic ATP turnover during contraction, although this just failed to reach statistical significance (P > 0.05). The results of the present study indicate that there is little difference in the muscle concentration of PCr estimated by the two methods, but that there are differences in the estimates of ATP, pH and lactate changes during contraction. This latter discrepancy may lead to greater estimates of ATP turnover being made as a result of MRS analysis.
Calcium paradox in guinea‐pig ventricular myocytes
Authors: Suleiman MS , Edmond JJ , Bulstrode GK
Published: December 2014

Measurement of the release of lactate dehydrogenase (LDH), the development of contracture and the exclusion of the dye Trypan Blue were made in isolated guinea‐pig ventricular myocytes subjected to conditions that evoke a calcium paradox in intact heart. Incubation at 25 degrees C reduced the release of LDH associated with mechanical manipulation of the myocytes so that during prolonged incubation in media free of Ca2+ and Mg2+ ions at 25 degrees C little release of LDH occurred. A significant release of LDH into the incubation media was provoked on repletion of the divalent cations. This release was accompanied by a marked decrease in the percentage of rod‐shaped myocytes and their ability to exclude Trypan Blue. The results provide evidence for the existence of the calcium paradox in isolated guinea‐pig ventricular myocytes as originally defined.
Cardiovascular and pancreatic endocrine responses to glucagon‐like peptide‐1(7–36) amide in the conscious calf
Authors: Edwards CM , Edwards AV , Bloom SR
Published: December 2014

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.
Nitric oxide in the control of submandibular gland function in the anaesthetized ferret
Authors: Tobin G , Edwards AV , Bloom SR , Ekstrom J
Published: December 2014

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.
Pituitary adenylyl cyclase‐activating polypeptides and vasoactive intestinal peptide inhibit bone resorption by isolated rabbit osteoclasts
Authors: Winding B , Wiltink A , Foged NT
Published: December 2014

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.
Mechanisms responsible for changes in abdominal vascular volume during sympathetic nerve stimulation in anaesthetized dogs
Authors: Noble BJ , Drinkhill MJ , Myers DS , Hainsworth R
Published: December 2014

This study was designed to determine the extent to which the decrease in volume of blood in the abdominal circulation in response to sympathetic stimulation was due to a passive effect of decreasing flow rather than active constriction of the capacitance vessels. In dogs anaesthetized with alpha‐chloralose (100 mg kg‐1 i.v.) the abdominal circulation was vascularly isolated and perfused either at constant flow or at constant pressure, and drained at constant pressure from the inferior vena cava. Changes in volume were determined by integration of the differences between inflow and outflow. Supramaximal stimulation of both splanchnic (sympathetic) nerves at 1 Hz decreased abdominal volume during constant pressure perfusion (active and passive components) by 3.04 +/ࢤ 0.58 ml kg‐1 and at constant flow (active responses only) by 2.30 +/ࢤ 0.49 ml kg‐1 (means +/ࢤ S.E.M.). The responses at 8 Hz were respectively 9.52 +/ࢤ 0.91 and 5.09 +/ࢤ 0.49 ml kg‐1. The proportion of the responses calculated to be passive at 1 and 8 Hz was 23 +/ࢤ 6.3 and 45 +/ࢤ 5.1%, respectively. These responses were almost identical to those induced by changing inflow by increasing the pump speed. Following ligation of the splenic pedicle, the responses during both constant pressure and constant flow were reduced by similar amounts, indicating that only the active response was affected. After ligation of the splenic pedicle, the proportion of the response calculated to be passive at 1 and 8 Hz increased to 44 +/ࢤ 8.0 and 62 +/ࢤ 3.7% respectively. These results indicate the importance of passive volume change in affecting abdominal volume, particularly following ligation of the splenic circulation.
Increased sensitization of the myofilaments in rat neonatal portal vein: a potential mechanism
Authors: Bruce L , Nixon GF
Published: December 2014

The contractile regulation of neonatal smooth muscle was studied in rat neonatal portal vein. Strips of beta‐escin‐permeabilized portal vein from 3‐ to 5‐day‐old rats and 5‐ to 6‐week‐old rats were mounted on a ‘bubble chamber’ in calcium solutions buffered with 10 mM EGTA. Although the overall tension development was lower in neonatal muscle as expected, the calcium sensitivity of the neonatal permeabilized portal vein was significantly higher than in developed portal vein. Endothelin‐1‐induced sensitization of the myofilaments was investigated. Endothelin‐1 produced an increase in tension of permeabilized neonatal portal vein in a calcium solution buffered with 10 mM EGTA. This sensitization was proportionally higher in neonatal than in developed smooth muscle, despite similar initial submaximal calcium contractions. GTP gamma S‐induced calcium sensitization was also proportionally higher in neonatal permeabilized strips than in fully developed smooth muscle. These changes may be due to alterations in the intracellular signalling pathways which mediate calcium sensitization in smooth muscle. As some endothelin‐1‐mediated responses are known to occur via activation of the heterotrimeric GTP‐binding protein, Gq, the levels of protein expression of Gq alpha were studied. In membrane preparations from neonatal rat portal vein the expression of Gq alpha was significantly higher than in portal vein membrane preparations loaded with equal protein from 5‐ to 6‐week‐old rats. In conclusion, agonist‐induced sensitization of the myofilaments was higher in neonatal rat portal vein than in fully developed portal vein. This difference is the result of changes in intracellular signalling and may be partly produced by the greater expression of Gq alpha observed in neonatal portal vein.