The effects of an acetylcholine analogue, carbachol (CCh), and a purified irreversible nicotinic antagonist, α-bungarotoxin (BTX), on the frequency of the miniature endplate potentials (mEPPs) at the neuromuscular junction of the frog were tested at 20 and 10¡C. CCh (5 ± 10-6 m) reduced the frequency of mEPPs to about 60 %; this reduction was not affected by 1 ± 10-7 g ml-1 BTX. BTX also reversibly decreased the mEPP frequency by 40 %, but not in the presence of CCh or in Ringer solution with 0 or 8 mM Ca2+. The present data show that BTX, which inhibits a class of nicotinic ACh receptors, does not block the decrease of mEPP frequency evoked by CCh and can itself suppress the frequency of spontaneous quantal release.

The study was designed to develop an improved technique for perfusing the isolated caudal lobe of sheep liver. Twenty caudal lobes were perfused for 3-4 h, in a non-recirculating mode, with Krebs-Henseleit bicarbonate buffer. The perfusion system was designed to give a constant flow. The hepatic viability and functional normality of the perfused lobe were assessed by measuring the perfusion flow rate, pH, K+ efflux, O2 uptake, substrate uptake, gluconeogenesis from propionate and amino acids, and ureagenesis from ammonia and amino acids. Liver tissue was sampled for histological examination, as well as for the determination of liver glycogen and wet : dry weight ratio. The perfusion flow rate and pH were both stable throughout the perfusion. The potassium concentration in the effluent perfusate did not increase during the perfusion, suggesting that there was no loss of viability or hypoxia. The perfused lobe extracted more than 50 % of the O2 supply. The rate of oxygen consumption was comparable to the rate reported in vivo. The initial glycogen content was reduced by about 40 % after 4 h perfusion. The wet : dry weight ratio was 3.6, consistent with the absence of tissue oedema. Urea production was stimulated when NH4Cl (0.3 mM) was added to the medium but there was no significant increase in urea release when alanine (0.15 mM), glutamine (0.2 mM) or lysine (0.2 mM) was added. Urea production, however, increased by about 171 % when a physiological mixture of amino acids was added. Propionate (0.5 mM), alanine and glutamine stimulated glucose production but not lysine or the complete amino acid mixture. Glutamine release was lower than that reported in the rat liver. Changing the direction of flow also revealed an apparent difference between livers from sheep and rats in their metabolism of ammonia. The improved technique offers a simple practical and inexpensive approach to many problems in ruminant physiology and nutritional biochemistry. Experimental Physiology (2000) 85.5, 469-478.

Brain-derived calcium-binding protein S100 induces apoptosis in a significant fraction of rat phaeochromocytoma (PC12) cells. We used single cell techniques (patch clamp, videomicroscopy and immunocytochemistry) to clarify some of the specific aspects of S100-induced apoptosis, the modality(ies) of early intracellular Ca2+ concentration increase and the expression of some classes of genes (c-fos, c-jun, bax, bcl-x, p-15, p-21) known to be implicated in apoptosis of different cells. The results show that S100: (1) causes an increase of [Ca2+]i due to an increased conductance of L-type Ca2+ channels; (2) induces a sustained increase of the Fos levels which is evident since the first time point tested (3 h) and remains elevated until to the last time point (72 h). All these data suggest that S100-derived apoptosis in PC12 cells may be the consequence of a system involving an increase in L-type Ca2+ channel conductance with consequent [Ca2+]i increase which up-regulates, directly or indirectly, the expression of Fos. Experimental Physiology (2000) 85.3, 243-253.

The ability to see the world around us is an immediate and striking example of the abilities of the nervous system, and perhaps for this reason, vision is one of the most intensively studied aspects of brain function (Hubel, 1995). This paper examines some of the earliest steps in vision occurring in the retina (Dowling, 1987; Rodieck, 1998).

A K+ channel activated by intracellular ATP has been observed in inside-out patches from rat atrial cells. The channel has a slope conductance of 130 ± 5 pS in symmetrical 140 mM K+ solution, and is almost independent of voltage over the range from -80 to +80 mV. There is no detectable inactivation during application of ATP over a few minutes. In the presence of 3 mM intracellular ATP, channel openings occur as bursts with a mean open time of 1.7 ms, a mean closed time of 0.4 ms, a mean burst duration of 18 ms and a mean burst interval of 41 ms. Kinetic analysis suggests that ATP mainly affects the burst duration and the burst interval of the channel. Based on the properties above, the channel differs from other known K+ channels in cardiac cells and may contribute to background K+ current.

he most popular approach for generating transgenic mammals is the direct injection of transgenes into one pronucleus of a fertilized oocyte. In the past 15 years microinjection has been successfully applied in laboratory as well as in farm animals. The frequency of transgenic founders, although highly different between the species, is efficient enough to render this technique applicable to a wide range of mammals. The expression levels and patterns of a transgene are initially influenced by the construction of the transgene. However, the overall phenotype of a transgenic organism is influenced by several genetic and environmental factors. Due to the features of this technique not all of the genetic factors can be experimentally controlled by the scientist. In this article we will emphasize some peculiarities which have to be taken into account for the successful performance of transgenesis by pronuclear microinjection

The dietary flavonol quercetin induces chloride secretion in rat intestine. To clarify the underlying mechanisms, experiments were performed in Ussing chambers with tissue from rat proximal and distal colon. Quercetin induced an increase in short-circuit current (Isc), which was largely independent of submucosal neurons, as it was not affected by the neurotoxin tetrodotoxin. The effect of quercetin was blocked by the calmodulin antagonists trifluoperazine and ophiobolin A and was diminished by a blocker of Ca2+ release from intracellular stores (TMB-8), whereas the muscarinic receptor antagonist atropine was ineffective. The quercetin-induced Isc was abolished in Ca2+-free solution. The flavonol was able to further increase Isc after maximal stimulation of the cAMP pathway by forskolin. The Isc increase by the flavonol was differently affected by two analogous phosphodiesterase inhibitors. Whereas 3-isobutyl-1-methylxanthine (IBMX) antagonized the effect of quercetin, 8-methoxymethyl-IBMX had no effect. Both phosphodiesterase inhibitors similarly influenced the Isc increase induced by forskolin. These results indicate that the chloride secretion induced by quercetin in rat colon depends on Ca2+ and calmodulin. The cAMP pathway and inhibition of phosphodiesterase appear not to be responsible for the secretory activity of the flavonol. Experimental Physiology (2000) 85.3, 255-261.

Previous studies have shown that central administration of GABA (gamma -aminobutyric acid), an inhibitory neurotransmitter, preferentially reduces hindquarters and carotid vascular resistances but not renal and coeliac vascular resistances in conscious rats. This study tested the hypothesis that these preferential actions of central GABA receptors are related to differences between vessels in resting autonomic vascular tone in freely moving rats. Rats were chronically implanted with intracisternal cannulas and/or electromagnetic probes to measure regional blood flows. In response to GABA administration, the changes in vascular resistance (arterial blood pressure/regional blood flow) of the hindquarters (n = 23) and carotid (n = 12) vascular beds were significantly and negatively correlated with basal vascular resistance. No such relationship was found for the renal (n = 21), coeliac (n = 13) and superior mesenteric (n = 23) vascular beds. This finding indicates that the responsiveness to GABA of brainstem pathways controlling the hindquarters and carotid vascular beds co-varies with resting resistance in hindquarters and carotid vessels. A similar analysis was performed, correlating the ongoing vascular resistance of each vessel with its response to ganglionic blockade by chlorisondamine. In this case, a significant negative correlation was also found for the hindquarters (n = 26) and carotid (n = 15) vascular beds, but not for the coeliac (n = 17) or superior mesenteric (n = 19) vessels. Together, these findings suggest that central GABA receptors accessible from the cisterna magna preferentially affect two vascular beds which, in the freely moving rat, show resting autonomic vascular tone. Experimental Physiology (2000) 85.5, 479-485.

System A-mediated amino acid transport, activation of different steps of signal transduction and involvement of different isoforms of protein kinase C (PKC) have been investigated in chick embryo hepatocytes after epidermal growth factor (EGF) stimulation. EGF rapidly (10 min) increased the rate of aminoisobutyric acid (AIB) uptake in chick embryo hepatocytes freshly isolated on the 19th day of embryonic life, while no change was detectable at other embryonal stages. The growth factor stimulation was abolished by PKC and tyrosine kinase inhibitors and was mimicked by 4-phorbol-12-myristate-13-acetate, dimethyl-2 (PMA). EGF treatment did not modify the phosphorylation of the γ isoform of phospholipase C (PLC-γ), and inositol trisphosphate (IP3) and intracellular calcium levels, but it induced an increase in PKC activity. Our data show that EGF regulates amino acid uptake, via PKC and without PLC-γ activation, only in the last period of chick embryo hepatocyte development. The effects of growth factor on PKC activity suggest the involvement of PKC-α and -ε isoforms in EGF modulation of amino acid transport.

Deuterium oxide (D2O) is known to cause a negative inotropic effect in muscle although the mechanisms associated with this response in cardiac muscle are not well understood. We studied the effects of D2O in single rat ventricular myocytes in order to characterise the mechanisms associated with its negative inotropic effect and to assess its possible use as an acute modulator of microtubules. D2O rapidly reduced the magnitude of contraction in rat ventricular myocytes, and there was some recovery of contraction in the presence of D2O. Colchicine, an agent known to depolymerise microtubules, did not modify the effect of D2O. D2O decreased the L-type Ca2+ current (ICa), measured under whole cell and perforated patch clamp conditions. Slowing of the time to peak and a delay in inactivation of ICa were observed. Intracellular calcium ([Ca2+]i) and sodium ([Na+]i) were measured using the fluorescent indicators fura-2 and SBFI, respectively. The fall in contraction upon exposure to D2O was not associated with a fall in the [Ca2+]i transient; this response is indicative of a reduction in myofilament Ca2+ sensitivity. Both the [Ca2+]i transient and [Na+]i increased during the partial recovery of contraction in the presence of D2O. We conclude that a decrease in the myofilament sensitivity for Ca2+ and a reduction in Ca2+ influx via ICa are principally responsible for the negative inotropic effect of D2O in cardiac muscle. We found no evidence to explain the negative inotropic effect of D2O in terms of microtubule proliferation. In addition we suggest that acute application of D2O is not a useful procedure for the investigation of the role of microtubules in excitation-contraction coupling in cardiac muscle.

The development of gene targeting technology, the exchange of an endogenous allele of a target gene for a mutated copy via homologous recombination, and the application of this technique to murine embryonic stem cells has made it possible to alter the germ-line of mice in a predetermined way. Gene targeting has enabled researchers to generate mouse strains with defined mutations in their genome allowing the analysis of gene function in vivo. This review presents the essential tools and methodologies used for gene targeting that have been developed over the past decade. Special emphasis has been laid on the available embryonic stem cell lines and the importance of the genetic background. Also, the state-of-the art of gene targeting approaches in species other than mice will be discussed.

The objective of this study was to investigate the effect of Na+-H+ exchange (NHE) and HCO3--Na+ symport inhibition on the development of rigor contracture. Freshly isolated adult rat cardiomyocytes were subjected to 60 min metabolic inhibition (MI) and 5 min re-energization (Rx). The effects of perfusion of HCO3- or HCO3--free buffer with or without the NHE inhibitor HOE642 (7 µM) were investigated during MI and Rx. In HCO3--free conditions, HOE642 reduced the percentage of cells developing rigor during MI from 79 ± 1 % to 40 ± 4 % (P < 0.001) without modifying the time at which rigor appeared. This resulted in a 30 % reduction of hypercontracture during Rx (P < 0.01). The presence of HCO3- abolished the protective effect of HOE642 against rigor. Cells that had developed rigor underwent hypercontracture during Rx independently of treatment allocation. Ratiofluorescence measurement demonstrated that the rise in cytosolic Ca2+ (fura-2) occurred only after the onset of rigor, and was not influenced by HOE642. NHE inhibition did not modify Na+ rise (SBFI) during MI, but exaggerated the initial fall of intracellular pH (BCEFC). In conclusion, HOE642 has a protective effect against rigor during energy deprivation, but only when HCO3--dependent transporters are inhibited. This effect is independent of changes in cytosolic Na+ or Ca2+ concentrations.

The response of pulmonary arteries to hypoxia varies as a function of vessel diameter. Small intrapulmonary resistance arteries are thought to be the main site of hypoxic pulmonary vasoconstriction (HPV), with hypoxia causing minimal contraction or even dilatation in large, conduit vessels. This has been proposed to reflect a differential distribution of morphologically and electrophysiologically distinct pulmonary artery smooth muscle (PASM) cells. We investigated longitudinal heterogeneity in smooth muscle cells isolated from five regions of the rabbit pulmonary vasculature and could find no evidence of morphological heterogeneity at the level of the light microscope. PASM cells from main (8 mm outer diameter) and branch (5 mm) arteries and large ( 400 m) intrapulmonary arteries (IPA) were similar in shape and size, as indicated by cell capacitance (25 pF). PASM cells from medium (200-400 m) and small ( 200 m) IPA were significantly smaller (15 pF), but had the same classical spindle shape. Cells from all five regions also had similar resting membrane potentials and displayed voltage-activated K+ currents of similar amplitude when recorded in standard physiological solution. Longitudinal heterogeneity in K+ current became apparent when tetraethylammonium ions (TEA; 10 mM) and glibenclamide (10 M) were added. The remaining delayed rectifier current (IK(V)) doubled in amplitude upon moving down the pulmonary arterial tree from the main artery (9 pA pF-1 at 40 mV) to the large IPA (17 pA pF-1), but remained constant throughout the intrapulmonary vasculature. The O2-sensitive, non-inactivating K+ current (IK(N)) showed a similar trend, but was significantly reduced in the smallest IPA, where its amplitude was comparable with the main artery. Thus the IK(N)/IK(V) ratio was relatively constant, at around 0.14, from the main pulmonary artery to medium IPA, but fell by 50 % in the smallest vessels. The amplitude of the TEA-sensitive K+ current was similar (16 pA pF-1 at 40 mV) at all levels of the pulmonary arterial tree, except in the medium sized vessels where it was 50 % smaller. These variations in K+ current expression correlate with reported variations in sensitivity to hypoxia and may contribute to the regional heterogeneity of HPV in the rabbit lung. Experimental Physiology (2000) 85.5, 487-496.

We investigated the permeability of Cs+ and Na+ through various ion channels in rat atrial myocytes using the whole-cell voltage-clamp technique. With isotonic CsCl (140 mM) on both sides of the membrane and nominally [Ca2+]o-free conditions, depolarising clamp pulses induced an increase of outward currents which showed a biphasic time course. Repolarisation to the holding potential induced inward tail currents. With isotonic NaCl, depolarisation also induced outward currents which showed a monotonic decay, but inward tail currents were not observed. Both in NaCl and CsCl, currents were hardly affected by TEA (10 mM), 4-AP (5 mM) and DIDS (100 µM). Nicardipine (1 M) almost completely blocked time-dependent outward currents in isotonic NaCl solution, leaving only time-independent currents which showed linear I-V relationship. In isotonic CsCl conditions, nicardipine blocked outward current considerably, but there still remained time-dependent outward currents and inward tail currents. Addition of E-4031 (2-20 M) which is known as a specific blocker of the rapidly activating delayed rectifier K+ current (IKr) completely blocked these time-dependent outward and inward currents, leaving only a time-independent current. Time-independent currents recorded in the presence of nicardipine and E-4031 were inhibited by GdCl3, which is known to block non-selective cation (NSC) currents. From these results, it was suggested that NSC current in atrial myocytes can be investigated in isotonic Cs+ or Na+ solution in the presence of Ca2+ channel and IKr blockers.

Permeabilized patch whole-cell voltage clamp methods were used to investigate the effects of isoprenaline (ISO) on total delayed rectifier potassium current, iK, in rabbit sino-atrial (SA) node pacemaker cells; total iK is composed of the rapidly activating iKr and the slowly activating iKs, but predominantly iKr in this species. ISO (20 nM) increased the amplitude of total iK and caused a negative shift of approximately 10 mV in the activation curve for iK, both in the absence and in the presence of 300 nM nisoldipine to block the L-type Ca2+ current, iCa,L. The same concentration (20 nM) of ISO increased the spontaneous pacemaker rate of SA node pacemaker cells by 16 %. In addition to increasing the amplitude of iK, ISO (20-50 nM) also increased the rate of deactivation of this current. The stimulation of iK by ISO was reversed by 10 µM H-89, a selective protein kinase A inhibitor, but not by 200 nM bisindolymaleimide I, a selective protein kinase C inhibitor. It therefore appears that the mechanisms by which β-adrenoceptor agonists increase pacemaking rate in sinoatrial node pacemaker cells include an increase in the rate of deactivation of iK in addition to the well-documented augmentation of iCa,L and the positive shift of the activation curve for the hyperpolarization-activated inward current, if. The observations are also consistent with a role for protein kinase A in the stimulation of iK by ISO in SA node cells.

Current applications of gene transfer in farm animals include the improvement of product quality and quantity, disease resistance, the production of valuable proteins in the mammary gland or other organs, the genetic modification of pigs for xenotransplantation and the generation of new animal models in cases where rodent models are not sufficient for studying the problem under evaluation. Although DNA microinjection into pronuclei of zygotes from various farm animal species has happened since 1985, the efficiency of this method is low. Further drawbacks are related to the random integration process which may cause mosaicism, insertional mutations and varying expression due to position effects. Sperm-mediated gene transfer is not routinely established yet, although the mechanisms of binding and internalisation of DNA by sperm cells is becoming increasingly clearer. New protocols for the use of retroviral vectors to infect metaphase II oocytes which are subsequently fertilised resulted in efficient production of transgenic cattle. In spite of extensive efforts to establish pluripotent stem cells from farm animal species, no germ-line competent cells have been reported in mammalian species other than mouse so far. However, recent success in cloning sheep, cattle, goats and pigs from cultured cells provides an alternative route for efficient and targeted genetic modifications of farm animals.

In chickens, we have shown that intestinal absorption of glucose via apical SGLT1 and basolateral GLUT2 transport systems is affected by dietary Na+; low-Na+ adapted birds show a dramatic reduction of glucose transporters in both membranes in the rectum, an intermediate response in the ileum and no effects in the jejunum. We have now studied the effect of resalination of low-Na+ adapted chickens on glucose kinetics across SGLT1 (using α -methyl-D-glucoside as substrate) and GLUT2 (using D-glucose) and on the specific binding of phlorizin and cytochalasin B, respectively. Twelve-week-old male Leghorn chickens were fed wheat and barley with drinking water containing either 150 mM NaCl (high-Na+ group) or 0.015 mM (low-Na+ group) for 14 days (serum aldosterone: 242 ± 6 pg ml-1 in the low-Na+ and 46 ± 4 pg ml-1 in the high-Na+ group). On day 14, the low-Na+ group was either resalinated with an oral dose of NaCl (9 g (kg body wt)-1) or switched to the high-Na+ condition, for 1 week. Serum aldosterone measured 4 h, 1 day and 7 days after the change in NaCl intake fell to between 30 and 39 pg ml-1. The changes in apical α-methyl-D-glucoside and basolateral D-glucose transport observed in the ileum and rectum of low-Na+ adapted animals were completely reversed by resalination within 4 h of NaCl administration to the level of values observed for high-Na+ adapted birds. The good correlation between the α-methyl-D-glucoside and D-glucose Vmax and the SGLT1 and GLUT2 density, respectively, supports the view that the increase in apical and basolateral hexose transport found in the ileum and rectum of both groups of resalinated birds is due to an increase in the number of protein transporters. The rapid changes in the number of glucose transporters observed suggest that the target of the regulatory signal(s) involved are the mature enterocytes present in the villi rather than the developing enterocytes in the crypt.

Exposure of human leukocytes to 12 % and 6 % O2in vitro resulted in striking ultrastructural and morphological changes. These changes included the appearance of crater-like holes, a reduction in granular size and disruption of the cellular membrane. The implications of these findings is discussed. Experimental Physiology (2000) 85.3, 263-266.

Experiments were performed to investigate the nature of α- and β-adrenoceptors in blood vessels supplying the posterior capsule of the acutely inflamed rabbit knee joint, and results were compared to findings from previous experiments on the normal joint, to assess any alteration which may occur in the adrenoceptor profile due to the inflammation process. Electrical stimulation of the posterior articular nerve resulted in vasoconstriction which was reversed to vasodilatation by phentolamine and yohimbine. The dose-response curves to close intra-arterial injection of α-adrenoceptor agonists showed a rank-order potency of: adrenaline = phenylephrine = clonidine. The adrenaline dose-response curve was shifted to the right by administration of antagonists with a rank-order potency of: phentolamine = yohimbine = prazosin. At this stage of the experiments there was an equal response of α1- and α2-adrenoceptors in blood vessels of the acutely inflamed rabbit knee joint. In another group of animals the neurally mediated vasodilatation, which appeared after administration of phentolamine, was completely blocked by propranolol, and was reduced by about 50 % by atenolol. The dose-response curves to close intra-arterial injection of β-adrenoceptor agonists showed a rank-order potency of: isoprenaline > salbutamol = dobutamine. The isoprenaline dose-response curve was shifted to the right by administration of antagonists with a rank-order potency of: propranolol > atenolol. These experiments also showed an almost equal response of β1- and β2-adrenoceptors in blood vessels of the acutely inflamed rabbit knee joint. Overall, compared to previous experiments on the normal joint in which α2- and β1-adrenoceptor responses predominated, acute inflammation resulted in a shift from α2- towards α1- and from β1- towards β2-adrenoceptor responses. Experimental Physiology (2000) 85.3, 267-273.

Increasingly sophisticated and precise molecular genetic tools are applied to mice in order to study the cellular mechanisms underlying higher brain functions, including learning and memory. However, several studies have produced unclear or conflicting results. One reason for this is that performance in the behavioural tests used to assess learning and memory is influenced by various non-cognitive phenomena and can thus easily be affected by mutations through mechanisms unrelated to memory function. We conducted principal component analysis on data from 3003 mice tested using a standardized protocol to demonstrate this for the Morris swimming navigation test, one of the most widely used paradigms to assess memory and hippocampal function. In addition, we present a meta-analysis showing that genetic background and environment alone produce sufficient variation to span the range of most, if not all, behavioural variables and can thus easily mask or fake mutation effects if genetic studies are not designed properly. We suggest that the chance of obtaining useful results is maximized if behavioural deficits are differentiated by combining complementary behavioural protocols and by analysing multiple complementary parameters in each of them. Mutation effects must be contrasted statistically against the influences of genetic background and environment. In many situations, this is most efficiently achieved if (i) mutations are backcrossed to and maintained in one or (preferably) two well-characterized, commonly available inbred strains and (ii) if mutant and wild-type littermates are analysed on a hybrid or mixed genetic background, that is in F1 or F2 generations derived from the inbred stocks