Biphasic short-circuit current response to noradrenaline mediated by Ca2+ and cAMP in cultured rat epididymal epithelium

A study was carried out to investigate the short-circuit current (I _sc) response to noradrenaline (NA) and the signal transduction mechanisms involved in cultured rat cauda epididymal epithelium. In normal Krebs-Henseleit solution, NA (10 mol · l^–1) added basolaterally elicited a biphasic I _sc response consisting of a transient spike followed by a second sustained response. The biphasic response was almost abolished by removing ambient Cl^–. Preloading the tissues witha cell-permeant Ca²⁺ chelator, 1,2-bis(2-aminophenoxy) eth-ane-N,N,N,N,-tetraacetic acid acetoxymethyl ester (BAPTA/AM), or pretreating them with thapsigargin (Tg), a microsomal adenosine triphosphatase inhibitor abolished the initial spike in the I _sc response to NA, but had little effect on the second component. Pretreating the tissues with a non-selective -antagonist, nadolol, reduced the second I _sc response in a dose-dependent fashion but the initial spike was not affected. Microfluorimetric studies showed that NA (100 mol · l^–1) elicited single Ca²⁺ spikes in isolated epididymal cells, which could be abolished by prior treatment with Tg. Biochemical assays showed that NA (10 mol · l^–1) increased intracellular cyclic adenosine monophosphate concentration ([cAMP]_i) and the response was abolished by prior treatment with nadolol (50 mol · l^–1). The results showed that NA elicited a biphasic I _sc response mediated by a rise in intracellular Ca²⁺ concentration ([Ca²⁺]_i) followed by a rise in [cAMP]_i. The Ca²⁺-mediated I _sc response had a faster onset and more transient action than the cAMP counterpart. It is suggested that NA released from noradrenergic nerve endings regulates transepithelial Cl^– secretion in the epididymis thereby providing the specialized millieu vital for sperm storage and maturation.     

Molecular specificity of the tubular resorption of “acidic” amino acids

Single sections of superficial proximal convolutions of rat kidney were microperfused in vivo and in situ. The perfusion fluids contained radioactively labelledl- ord-aspartate,l-glutamate,l-pyroglutamate, or N-methyl-d-aspartate.l--Carboxyglutamate as well as the other amino acids were added in the unlabelled from. Results.l- andd-Aspartate (0.073 mmol·1^–1) are quickly resorbed at about the same rate.d-Aspartate resorption was blocked byl-aspartate (5 mmol·1^–1) but not by -alanine (5 mmol·1^–1).l-Aspartate resorption was inhibited byl-glutamate (2 mmol·1^–1) but not byd-glutamate,l-asparagine,l-phenylalanine or by succinate (2 mmol·1^–1, each). The fast resorption ofl-glutamate (0.073 mmol·1^–1) was blocked byd-aspartate,l-cysteate (2 mmol·1^–1), but not by 3-mercaptopicolinic acid (0.15 mmol·1^–1),l-glutamine, 2-oxoglutarate, taurine, N-methyl-l-glutamate or kainic acid (2 mmol·1^–1, each).l--Carboxyglutamate (0.66 mmol·1^–1) and N-methyl-d-aspartate (2mol·1^–1) were found to be resorbed only at an extremely small rate.l-pyroglutamate (0.076 mmol·1^–1) resorption was not influenced byl-glutamate (1 mmol·1^–1). Fractional excretion of -carboxyglutamate was 7–25% (l-from) or 45–70% (d-form) at an artificially elevated plasma level of 12mol·1^–1.It is concluded thatl- andd-aspartate,l-glutamate,l-cysteate and, to a much smaller extent,l--carboxyglutamate, are accepted by the tubular resorption mechanism highly specific for acidic amino acids. N-Substitution, the amidation of the - or -carboxyl group, or the removal of the -amino moiety almost completely abolish the ability of such compounds to be resorbed via this carrier; N-methylated or -carboxylated derivatives of acidic amino acids are not resorbed at all from the proximal tubule. The resorption of glutamate, but not of aspartate, is highly stereospecific.Parts of this work were presented at meetings of the German Physiological Society in 1978 [28] and of the Gesellschaft für Nephrologie in 1980 [29] as well as at the VIIIth International Congress of Nephrology in Athens in 1981 [26]with technical assistance of Angelika Ascher and Gertaud Vetter     

Interactions between chromosomal omnipotent suppressors and extrachromosomal effectors in Saccharomyces cerevisiae

Summary Chromosomal omnipotent suppressor mutations recovered in ⁺ strains of Saccharomyces cerevisiae were brought into ^– cytoplasm. SUP46, SUP138 and SUP139 acted as dominant omnipotent suppressors in the ^– cytoplasm though their suppressor activity was substantially reduced. SUP46 and SUP138 conferred recessive thermosensitivity and antibiotic sensitivity in ^– cytoplasm as in ⁺ cytoplasm. On the other hand, sup111 through sup115, which acted as recessive omnipotent suppressors in the ⁺ cytoplasm, manifested no, or very low, suppressor activity in the ^– cytoplasm. They, however, still enhanced the efficiency of the SUP29 tRNA suppressor in ^– cytoplasm. A multicopy plasmid carrying the wild-type SUP35 gene enhanced the efficiency of sup111 in ^– cytoplasm.     

Overshoot in <Emphasis Type="Italic">V̇</Emphasis>O<Subscript>2</Subscript> following the onset of moderate-intensity cycle exercise in trained cyclists

We have previously observed that following the onset of moderate intensity cycle ergometry, the pulmonary O₂ uptake (O₂) in trained cyclists often does not increase towards its steady-state value with the typical mono-exponential characteristics; rather, there is a transient overshoot. The purpose of this study was to systematically examine this phenomenon by comparing the O₂responses to two moderate-intensity work rates and one high-intensity work rate in trained and untrained subjects. Following a ramp exercise test to the limit of tolerance for the determination of the gas exchange threshold (GET) and O_2peak, seven trained cyclists [mean (SD); O_2peak 66.6 (2.5) ml·kg^–1·min^–1] and eight sedentary subjects [O_2peak 42.9 (5.1) ml·kg^–1·min^–1] completed six step transitions from baseline cycling to work rates requiring 60% and 80% GET and three step transitions from baseline cycling to a work rate requiring 50% of the difference between GET and O_2peak (50%). O₂ was measured breath-by-breath and modelled using standard techniques. The sedentary subjects did not overshoot the steady-state O₂ at any intensity. At 60% GET, six of the seven cyclists overshot the steady-state O₂ [by an integral volume of 164 (44) ml between ~45 and 125 s]. At 80% GET, four of the seven cyclists overshot the steady-state O₂ [by an integral volume of 185 (92) ml between ~55 and 140 s]. None of the cyclists showed an overshoot at 50%. These results indicate that trained cyclists evidence an overshoot in O₂ before steady-state is reached in the transition to moderate-intensity exercise. The mechanism(s) responsible for this effect remains to be elucidated, as does whether the overshoot confers any functional or performance benefit to the trained cyclist.     

Assessment of plasma opioid peptides, β-endorphin and met-enkephalin,at the end of an international nordic ski race

Summary Plasma met-enkephalin, -endorphin, cortisol and lactic acid concentrations were measured in seventeen volunteer male subjects at rest and after a long-distance nordic ski race. Immediately after the race, mean plasma met-enkephalin did not show any significant change, but significant rises in -endorphin, cortisol and lactic acid were noted in all skiers. The change in -endorphin with exercise was significantly related to the change in cortisol (r=0.68;p<0.001) and to the change in plasma lactic acid (r=0.60;p<0.001). Furthermore, the experienced skiers training over 150 km · week^–1 of nordic ski had significantly faster skiing times in this event and showed greater -endorphin, cortisol and lactic acid levels than the recreational skiers who trained for 20 km · week^–1. Our results imply that the changes in plasma -endorphin depend on the intensity of exercise. However the significance of higher levels of skiing training or previous nordic ski experience in the release of -endorphin is expected and cannot be excluded.     

Serum contains a potent factor that decreases β-adrenergic receptor-stimulated L-type Ca2+ current in cardiac myocytes

L-type Ca²⁺ current (I _Ca) was measured in cultured atrial myocytes from hearts of adult guinea-pigs using whole-cell voltage clamp. Potentiation of I _Ca induced by -adrenergic stimulation (isoprenaline 2· 10^–7 M) could be completely antagonized by diluted sera (1100 v/v). Half-maximal inhibition of -receptorstimulated I _Ca occurred at about 11000. Basal I _Ca was not affected by serum. Atropine in a concentration (10^–6M) that completely antagonized the anti-adrenergic effect of acetylcholine (ACh, 2·10^–6 M) did not interfere with the effect of serum. In cells dialysed with cyclic adenosine monophosphate (cAMP)-containing (10^–4 M) pipette solution, potentiated I _Ca was insensitive to both ACh and serum. Preincubation of the myocytes with pertussis toxin almost completely abolished the anti-adrenergic effects of both ACh and serum. The potency of serum was not reduced by dialysis. It is concluded that serum contains a factor which, like ACh, inhibits -receptor-stimulated adenylyl cyclase via G_iprotein.A preliminary report of this work has appeared in abstract form [11]     

Sodium current in single myocardial mouse cells

Morphologically intact single myocardial cells of the adult mouse show a length of 132±20 m, a width of 21±5 , and a height of 10±4 m (all mean ± SD) and are brick-like in shape. A one suction pipette method is used for voltage clamp of those single cells. The determined time constant of capacitive current =35±14 s is very short. Series resistancer _s, membrane resistancer _m, and membrane capacityc _m are calculated to be 192±48 k, 6.1±1.1 M, and 186±92 pF (all mean ± SD), respectively. Assuming the specific unit membrane capacitance of 1 F/cm², a total membrane area of 1.86×10^–4 cm² is determined yielding a specific membrane resistanceR _m of 1,134 cm². Settling time of voltage clamp is 30 s. TTX-block of sodium current is described by 1:1 binding with aK _D value of 1.4×10^–6M. Using a reduced extracellular sodium concentration the maximum Na current is between 25 and 40 nA at voltages between –40 and –30 mV. Currents of between +20 and +30 mV reverse in an outward direction. Inward currents are approximated by a m³h model. The time constant of activation decreases from 0.7 ms at –60 mV to 0.12 ms at +20 mV. The time constant of inactivation falls from 9.1 ms at –60 mV to 0.6 ms at +20 mV.Steady state inactivationh is characterized by the half maximum valueV _H=–76.1±4.3 mV and the slope parameters=–6.3±1.1 mV (mean ± SD). A prepulse duration of 500 ms is essential for real steady state inactivation. Steady state activationm and inactivationh overlap each other defining a maximum window current at –65 mV.     

Adjustment of metabolism,catecholamines and β-adrenoceptors to 90 min of cycle ergometry

Adrenaline infusion of 0.1 g · kg^–1 · min^–1 in healthy volunteers results in an increase of hepatic glucose production, an increase of the absolute number of occupied -adrenoceptors and specific changes in metabolism. To compare these effects with the changes induced by an endogenous catecholamine release, we investigated healthy volunteers during cycle ergometry. After fasting at least 14 h seven healthy subjects exercised for 90 min at an intensity of 20% below their individual anaerobic threshold. The rate of glucose production as well as the turnover rates of alanine and leucine were calculated using stable isotope tracers. High and low affinity -adrenergic binding sites on lymphocytes were determined by an equilibrium binding assay with (–)¹²⁵ Iodocyanopindolol. After 90 min of cycling the rate of appearance of glucose increased significantly from means of 2.0 (SD 0.2) to 2.65 (SD 0.50) mg · kg^–1 · min^–1 with unchanged blood concentrations of glucose and lactate. The flux of the amino acids alanine and leucine decreased significantly from means of 0.91 (SD 0.21) to 0.62 (SD 0.14) mg · kg^–1 · min^–1 and from 0.40 (SD 0.05) to 0.32(SD 0.04) mg · kg^–1 · min^–1, respectively. The mean free fatty acid concentration increased significantly from 0.65 (SD 0.33) to 1.27 (SD 0.45) mmol · l^–1 during the endurance trial. The increase of glucose turnover and the decrease of amino acid flux point to a metabolic shift towards enhanced utilization of free fatty acids. Adrenaline and noradrenaline concentrations showed a moderate but significant increase from means of 0.61 (SD 0.20) to 0.99 (SD 0.36) nmol · l^–1 and from 2.27 (SD 0.75) to 3.46 (SD 0.38) nmol · 1^–1, respectively. The number of high affinity -adrenergic binding sites per cell (-adrenoceptors) nearly doubled from 770 (SD 130) to 1490 (SD 150) during 90 min of cycling. The observed endogenous plasma catecholamine concentrations were not sufficient to change significantly the relative receptor occupancy. This would seem to indicate that the aerobic exercise induced effects depended more on the absolute number of occupied -adrenoceptors than on their relative receptor occupancy. When compared to the results of the adrenaline infusion experiment the increases of the hepatic glucose production and the increase of -adrenoceptors were very similar in both groups despite ten times higher adrenaline plasma concentrations in the infusion group. This would seem to indicate that -adrenoceptors mediated effects do not correlate with catecholamine plasma concentrations.     

The “small” conductance chloride channel in the luminal membrane of the rectal gland of the dogfish (Squalus acanthias)

Besides the larger Cl^– channel with a single channel conductance of about 45 pS, a small channel was observed in the luminal membrane of the dogfish rectal gland [9]. In cell excised (inside out) patches with NaCl solution on both sides, the latter channel had a single channel conductance of 11±1 pS (n=21), and its current-voltage relationship was linear in the voltage range+90 to –90 mV. The open state probability increased moderately with negative clamp potentials. Ionic replacement studies revealed a high selectivity of Cl^– over gluconate, sulfate, and iodide, whereas bromide was permeable to some extent. Also the channel is impermeable for Na⁺. The Cl^– channel blocker 5-nitro-2-(3-phenylpropylamino)-benzoate did not affect this small conductance Cl^– channel. It can be concluded that the luminal membrane of stimulated rectal gland cells possesses two types of Cl^– channels, which differ markedly in their characteristics.Supported by Deutsche Forschungsgemeinschaft Gr 480/8 and by NSF and NIH grants to the MDIBL     

Intracellular pH regulation of human colonic crypt cells

In order to investigate the regulation of intracellular pH (pH_i) in freshly isolated human colonocytes, we have used a newly developed technique for the rapid isolation and covalent attachment of these cells to glass surfaces and microspectrofluorimetric measurement of the pH-sensitive fluorescence of 2,7-bis(carboxyethyl)-5(6)-carboxyfluorescein (BCECF)-loaded specimens in a perfusion chamber (37°C). In N-2-hydroxyethylpiperazine — N — 2 — ethanesulphonic — acid — (HEPES)-buffered Ringer solution (HBS) a baseline pH_i of 7.35±0.03 (mean ± SD; n=42) was found for human colonocytes and in HBS, NH₄Cl-prepulse-induced intracellular acidification in colonocytes is reversed rapidly by the ubiquitous amiloride-sensitive (1 mmol/l) Na⁺/H⁺ exchanger. Switching from HBS to HCO ₃ ^– buffered solution (BBS) led to a transient intracellular acidification (7.29±0.09), followed by a recovery to a final resting pH_i of 7.43±0.03. One-third of the acid extrusion in BBS is amiloridesensitive; the remaining two-thirds are caused by the dihydroderivative of 4,4-diisothiocyanatostilbene-2,2-disulphonic acid (H₂DIDS)-sensitive HCO ₃ ^– -dependent mechanisms. The functional activity of an acid-extruding Na⁺/HCO ₃ ^– cotransporter in human colonocytes was observed in response to the reintroduction of Na⁺ into amiloride-containing Na⁺/Cl^–-free BBS. In addition, the mechanism leading to alkalinization (7.56±0.05) in Cl^–-free BBS was identified as Na⁺-dependent Cl^–/HCO ₃ ^– exchange, by its H₂DIDS sensitivity and the specific requirement for Cl^– and Na⁺. The intrinsic buffering capacity ( _i) of the human colonocytes was calculated from pH changes induced by sequential NH₄Cl-loading steps during blockage of acid/base transporters. With _i=80 mmol · l^–1 · pH unit^–1 for the pH interval ranging from 6.9 to 7.1 (n=8) the colonocytes exhibited a relatively high intrinsic buffering in comparison with other cell types. In conclusion, the freshly isolated human colonocytes express a Na⁺/H⁺ exchanger, a Na⁺/HCO ₃ ^– cotransporter and a Na⁺-dependent Cl^–/HCO ₃ ^– exchanger, all of them likely to be involved in the regulation of pH homeostasis in vivo in the presence of widely varying extracellular conditions. Maintenance of a stable pH_i of human colonocytes seems to be facilitated by a comparatively high _i at physiological pH values.     

Renal tubular reabsorption of taurine,γ-aminobutyric acid (GABA) andβ-alanine studied by continuous microperfusion

Summary Renal tubular reabsorption of taurine, -aminobutyric acid (GABA), and -alanine was studied in vivo et situ by continuous microperfusion of single proximal tubules of the rat. In each case, reabsorption was much slower than that for other amino acids that have been studied. With a concentration of 0.1 mmol/l in initial perfusate, about 60% of initial load was reabsorbed over perfusion distance of 3 mm. Taurine reabsorption saturated with only 2.17 mmol/l in initial perfusate. Assuming simple two-parameter kinetics, upper limits for K _m of 0.54 mmol/l and forV _max of 0.59 pmol·cm^–1·s^–1 for tubular reabsorption of taurine were estimated. High (20 mmol/l) concentrations of taurine or -alanine in perfusate completely inhibited GABA reabsorption, butl-phenylalanine (20 mmol/l) had no significant effect. The results indicate that the three amino acids are reabsorbed slowly from the proximal tubule by what may be a common transport system. This system appears to have a high affinity but low capacity and to be different from other known renal tubular transport systems for amino acids.Supported by National Science Foundation Research Grant No. PCM 75-09918 and by grant No. 1740 of the Austrian Fonds zur Förderung der Wissenschaftlichen Forschung.     

Modulation of Nav1.5 by β1- and β3-subunit co-expression in mammalian cells

Cardiac sodium channels (Na_v1.5) comprise a pore-forming -subunit and auxiliary -subunits that modulate channel function. In the heart, 1 is expressed throughout the atria and ventricles, whilst 3 is present only in the ventricles and Purkinje fibers. In view of this expression pattern, we determined the effects of 3 and 1 co-expression alone, and in combination, on Na_v1.5 stably expressed in Chinese hamster ovary cells. The current/voltage relationship was shifted –5 mV with either 1 or 3 co-expression alone and –10 mV with co-expression of both 1 and 3. In addition, 3 and 1/3 co-expression accelerated macroscopic current decay. There were significant hyperpolarizing shifts in equilibrium gating relationships with co-expression of 1 and 3 alone and in combination. Co-expression of 1/3 together resulted in a greater hyperpolarizing shift in channel availability, and an increase in the slopes of equilibrium gating relationships. Co-expression of 3 and 1/3, but not 1, slowed recovery from inactivation at –90 mV. Development of inactivation at –70 and –50 mV was accelerated by -subunit co-expression alone and in combination. -Subunit co-expression also reduced the late Na current measured at 200 ms. In conclusion, -subunits modulate Na_v1.5 gating with important differences between co-expression of 1 and 3 alone and 1/3 together.     

Voltage-dependent noradrenergic modulation of ω-conotoxin-sensitive Ca2+ channels in human neuroblastoma IMR32 cells

High-threshold (HVA) Ca²⁺ channels of human neuroblastoma IMR32 cells were effectively inhibited by noradrenaline. At potentials between –20 mV and +10 mV, micromolar concentrations of noradrenaline induced a 50%–70% depression of HVA Ba²⁺ currents and a prolongation of their activation kinetics. Both effects were relieved at more positive voltages or by applying strong conditioning pre-pulses (facilitation). Facilitation restored the rapid activation of HVA channels and recruited about 80% of the noradrenaline-inhibited channels at rest. Re-inhibition of Ca²⁺ channels after facilitation was slow ( _r 36–45 ms) and voltage-independent between –30 mV and –90 mV. The inhibitory action of noradrenaline was dose-dependent (IC₅₀=84 nM), mediated by ₂-drenergic receptors and selective for -conotoxin-sensitive Ca²⁺ channels, which represent the majority of HVA channels expressed by IMR32 cells. The action of noradrenaline was mimicked by intracellular applications of GTP[S] and prevented by GDP[S] or by pre-incubation with pertussis toxin. The time course of noradrenaline inhibition measured during fast application (onset) and wash-out (offset) of the drug were independent of saturating agonist concentrations (10–50 M) and developed with mean time constants of 0.56 s ( _on) and 3.6 s ( _off) respectively. The data could be simulated by a kinetic model in which a G protein is assumed to modify directly the voltage-dependent gating of Ca²⁺ channels. Noradrenaline-modified channels are mostly inhibited at rest and can be recruited in a steep voltage-dependent manner with increasing voltages.     

β-Adrenoceptor-mediated depolarization of the resting membrane in guinea-pig papillary muscles: changes in intracellular Na+, K+ and Cl− activities

Effects of -adrenergc stimulation on the membrane potential and intracellular Na⁺, K⁺ and Cl^– activities were examined in isolated guinea-pig ventricular muscles using conventional and ion-selective elctrodes. Isoproterenol in concentrations of 30 nM–1 M produced a transient depolarization followed by a slight hyperpolarization in electrically stimulated or quiescent papillary muscles. The negative logarithm of the concentration producing 50% maximum effect (pD₂) for the membrane-depolarizing effect of isoproterenol was smaller than that for the positive inotropic effect, suggesting that a higher level of cAMP accumulation is required to produce the transient depolarization. Whereas the isoproterenol(1 M)-induced depolarization was not blocked by tetrodotoxin (10 M), nifedipine (10 M), Cs⁺ (5 mM), Ba²⁺ (0.3 mM), amiloride (1 mM) or ouabain (10 M), it was significantly attenuated by anthracene-9-carboxylic acid (1 mM), a Cl^–-channel blocker. Intracellular K⁺ activity increased, whereas intracellular Na⁺ activity slightly decreased during the transient depolarization. Intracellular Cl^– activity significantly decreased during the isoproterenol-induced depolarization of the resting membrane. These results suggest that an inward current resulting from outward Cl^– movement, rather than inward Na⁺ movement, may be involved in the -adrenoceptor-mediated membrane depolarization.     

Go transduces GABAB-receptor modulation of N-type calcium channels in cultured dorsal root ganglion neurons

High-voltage-activated (HVA) calcium channel currents (I _Ba) were recorded from acutely replated cultured dorsal root ganglion (DRG) neurons. I _Ba was irreversibly inhibited by 56.9±2.7% by 1 M -conotoxin-GVIA (-CTx-GVIA), whereas the 1,4-dihydropyridine antagonist nicardipine was ineffective. The selective -aminobutyric acid_B (GABA_B) agonist, (–)-baclofen (50 M), inhibited the HVA I _Ba by 30.7±5.4%. Prior application of -CTx-GVIA completely occluded inhibition of the HVA I _Ba by (–)-baclofen, indicating that in this preparation (–)-baclofen inhibits N-type current. To investigate which G protein subtype was involved, cells were replated in the presence of anti-G protein antisera. Under these conditions the antibodies were shown to enter the cells through transient pores created during the replating procedure. Replating DRGs in the presence of anti-G_o antiserum, raised against the C-terminal decapeptide of the G _o subunit, reduced (–)-baclofen inhibition of the HVA I _Ba, whereas replating DRGs in the presence of the anti-G_i antiseram did not. Using anti-G _o antisera (12000) and confocal laser microscopy, G _o localisation was investigated in both unreplated and replated neurons. G _o immunoreactivity was observed at the plasma membrane, neurites, attachment plaques and perinuclear region, and was particularly pronounced at points of cell-to-cell contact. The plasma membrane G _o immunoreactivity was completely blocked by preincubation with the immunising G_o undecapeptide (1 g · ml^–1) for 1 h at 37° C. A similar treatment also blocked recognition of G _o in brain membranes on immunoblots. These results provide evidence that GABA_B inhibition of N-type calcium channels in acutely replated DRGs occurs via G _o.     

Ca2+ not cyclic AMP mediates the fluid secretory response to isoproterenol in the rat mandibular salivary gland: Whole-cell patch-clamp studies

We have performed whole-cell patch-clamp studies on dispersed seccretory cells of the rat mandibular gland to determine how -adrenergic stimulation causes fluid secretion. When the pipette contained a high K⁺ solution, the resting membrane potential averaged –33 mV±1.1 (SEM,n=34) and the clamped cell showed strong outward rectification. We monitored K⁺ and Cl^– currents for periods of 15 min by recording the currents needed to clamp the cell potential at 0 and –80 mV, respectively. Isoproterenol (1–2 mol/l) caused increases in the clamp current at 0 mV (the K⁺ current) and at –80 mV (the Cl^– current) in about 80% of cases, although the responses were variable in size and time-course; the responses were indistinguishable from those induced by acetylcholine or the Ca²⁺ ionophore, A23187. The -adrenergic antagonist, phentolamine (1–2 mol/l), had no effect on the response, but the -adrenergic antagonist, propranolol (10 mol/l), blocked it completely. The isoproterenol response could not be mimicked by application to either surface of the cell membrane, of cyclic AMP (100 mol/l), forskolin (1 or 20 mol/l) or cholera toxin (2.5 g/ml). However, increasing the Ca²⁺-chelating capacity of the pipette solution by raising its EGTA concentration from the customary 0.5 to 20 mmol/l, blocked the response to isoproterenol, suggesting that -adrenergic agonists activate Cl^– and K⁺ channels by raising cytosolic Ca²⁺. Since neomycin, which blocks phospholipase C, blocked the action of isoproterenol without impairing the cell responsiveness to A23187, it appears that isoproterenol, like muscarinic agonists, increased cytosolic Ca²⁺ via the phosphatidylinositol cycle.This project was supported by the National Health and Medical Research Council of Australia     

Effect of “flow anoxia” and “non flow anoxia” on the NAD/NADH redox state of the intact brain cortex of the cat

In the present study, we compared the nicotin-amide adenine dinucleotide (NAD) reducing potencies of flow anoxia and non flow anoxia in the cat brain cortex. In animals anaesthetized with alpha-D-glucochloralose flow anoxia and non flow anoxia were produced by ventilating for 2 and 25 min, respectively, with nitrogen gas. Following non flow anoxia, the brain cortices of dead animals were superfused with oxygen saturated artificial cerebrospinal fluid (mock CSF), and subsequently with CSF containing various concentrations (10^–3–10^–1 M) of potassium cyanide. NADH (reduced NAD) fluorescence of the brain cortex was measured through a cranial window with microscope fluororeflectometer. Ventilating the animals for 2 and 25 min with nitrogen gas increased cortical NADH fluorescence (NAD reduction) by 43.5±2.8% and 135.3±6.1%, respectively. Oxygen saturated CSF superfusion of the ischemic brain cortex restored the cortical NAD/NADH redox state to the preanocic level (oxidation of NADH). 10^–1 M cyanide, applied after superfusion of the brain cortex with oxygen saturated CSF resulted in comparable NAD reduction to that produced by non flow anoxia. On the basis of these findings it is suggested that non flow anoxia leads to much greater cortical NAD reduction than flow anoxia, because oxygen tension in the cortex may not fall to zero mm Hg during nitrogen anoxia lasting for 2 min. Besides this, a more pronounced substrate mobilization and acidosis may also contribute to the greater NAD reducing potency of non flow anoxia. Finally, since 10–15 min after the death of the animal the cerebral carbohydrate reserves are completely exhausted, and in our experiments non flow anoxia, reoxygenation of the ischemic brain cortex and inhibition of the cortical mitochondrial electron transport by cyanide (10^–1 M) resulted in comparable redox state changes (as far as their magnitude is concerned), it is concluded that the recorded changes in NADH fluorescence were of mitochondrial origin.     

β-Endorphin and adrenocorticotrophin after incremental exercise and marathon running-female responses

Investigations of exercise-induced increases in -endorphin, adrenocorticotropic hormone (ACTH) and cortisol concentration have been carried out mainly in men. Data concerning the female reaction are sparse and less clear. In a comparison between incremental exercise and marathon running 14 experienced female marathon runners volunteered to run to exhaustion according to an incremental treadmill protocol. They ran a marathon 4 weeks later. Blood was analysed for -endorphin, ACTH and cortisol concentration immediately prior to the laboratory treadmill test, 3, 30 and 60 min later, as well as prior to the marathon, after 60 min and 120 min of running and 3, 30 min, and 24 h after completion of the run. At each blood collection, lactate concentration, heart frequency and perceived exertion were determined. The mean marathon running time was 3.22 h. Baseline concentrations for -endorphin of 22 pmol · l^–1 before the marathon and 19 pmol · l^–1 before the treadmill exercise increased 1.4-fold 30 min after the marathon and 1.9-fold after the treadmill exercise; for ACTH the baseline of 4.7 and 4.0 pmol · l^–1 t was increased by 8.3- and 10.3-fold, respectively. Cortisol concentration rose exponentially from a baseline 17 g · dl^–1 and peaked at 2.2-fold 30 min after the run, when the maximal concentration also had been reached after the treadmill test, increasing 1.3-fold from a baseline of 21 g · dl^–1. The maximal values for cortisol concentration after both exercises differed from each other, while the maxima of ACTH and -endorphin concentrations were similar. The ACTH and -endorphin concentration declined more slowly during the recovery after the marathon than after the treadmill. Cortisol concentration was below baseline 24 h later. In comparison with men studied earlier, female marathon runners showed higher baseline concentrations and lesser increases in -endorphin and lower baseline concentrations and larger increases in ACTH concentration after both types of exercise. The delayed decrease in concentration of the hormones after the marathon was similar in male and female runners.     

Inhibition of ω-conotoxin-sensitive Ca2+ channel currents by internal Mg2+ in cultured rat cerebellar granule neurones

The effects of changing the intracellular concentrations of either free Mg²⁺ ions ([Mg²⁺]_i) or Mg²⁺-bound adenosine triphosphate ([Mg · ATP]_i) on Ca²⁺ channel currents were assessed in cultured rat cerebellar granule neurones using the whole-cell patch-clamp technique. Raising [Mg²⁺]_i from 0.06 mM to 1.0 mM inhibited Ca²⁺ channel currents by approximately 50%. The action of -conotoxin GVIA (-CgTX), a selective inhibitor of N-type Ca²⁺ channels was also investigated. With increasing [Mg²⁺]_i, the proportion of current irreversibly blocked by -CgTX was reduced, and was negligible (approximately 5 pA of current) in the presence of [Mg²⁺]_i values of 0.5 mM or greater. Block of the -CgTX-sensitive current accounted for the reduction in total current by concentrations of [Mg²⁺]_i to 0.5 mM. Raising [Mg²⁺]_i had no effect on the rate of decay of Ca²⁺ currents, but did produce a negative shift in current activation, possibly due to a non-specific interaction with negative surface charge. Altering [Mg · ATP]_i from 0.3 to 5.0 mM caused a twofold increase in the size of currents without affecting the proportion of current sensitive to -CgTX. [Mg²⁺]_i was also effective in inhibiting the Ca²⁺ channel current following potentiation by increasing [Mg · ATP]_i. These data suggest that -CgTX-sensitive current in these cells is selectively inhibited by internal Mg²⁺ whereas both -CgTX-sensitive and -resistant components of current are potentiated by internal Mg · ATP. The mechanism by which Mg²⁺ inhibits N-type channels is unclear, but may involve an open channel block.     

Influence of muscle fibre type and pedal rate on the V̇O2-work rate slope during ramp exercise

We hypothesised that the ratio between the increase in oxygen uptake and the increase in work rate (O₂/WR) during ramp cycle exercise would be significantly related to the percentage type II muscle fibres at work rates above the gas exchange threshold (GET) where type II fibres are presumed to be active. We further hypothesised that ramp exercise at higher pedal rates, which would be expected to increase the proportional contribution of type II fibres to the total power delivered, would increase the O₂/WR slope at work rates above the GET. Fourteen healthy subjects [four female; mean (SD): age 25 (3) years, body mass 74.3 (15.1) kg] performed a ramp exercise test to exhaustion (25 W min^–1) at a pedal rate of 75 rev min^–1, and consented to a muscle biopsy of the vastus lateralis. Eleven of the subjects also performed two further ramp tests at pedal rates of 35 and 115 rev min^–1. The O₂/WR slope for exercise <GET (S ₁) was significantly correlated with O₂ peak in ml kg^–1 min^–1 (r=0.60; P<0.05), whereas the O₂/WR slope for exercise >GET (S ₂) was significantly correlated to percentage type II fibres (r=0.54; P=0.05). The ratio between the O₂/WR slopes for exercise above and below the GET (S ₂/S ₁) was significantly greater at the pedal rate of 115 rev min^–1 [1.22 (0.09)] compared to pedal rates of 35 rev min^–1 [0.96 (0.02)] and 75 rev min^–1 [1.09 (0.05), (P<0.05)]. The greater increase in S ₂ relative to S ₁ in subjects (1) with a high percentage type II fibres, and (2) at a high pedal rate, suggests that a greater recruitment of type II fibres contributes in some manner to the xs O₂ observed during ramp exercise.