Showing papers in "Pflügers Archiv: European Journal of Physiology in 1981"

Improved patch-clamp techniques for high-resolution current recording from cells and cell-free membrane patches.

Abstract: 1. The extracellular patch clamp method, which first allowed the detection of single channel currents in biological membranes, has been further refined to enable higher current resolution, direct membrane patch potential control, and physical isolation of membrane patches. 2. A description of a convenient method for the fabrication of patch recording pipettes is given together with procedures followed to achieve giga-seals i.e. pipette-membrane seals with resistances of 10(9) - 10(11) omega. 3. The basic patch clamp recording circuit, and designs for improved frequency response are described along with the present limitations in recording the currents from single channels. 4. Procedures for preparation and recording from three representative cell types are given. Some properties of single acetylcholine-activated channels in muscle membrane are described to illustrate the improved current and time resolution achieved with giga-seals. 5. A description is given of the various ways that patches of membrane can be physically isolated from cells. This isolation enables the recording of single channel currents with well-defined solutions on both sides of the membrane. Two types of isolated cell-free patch configurations can be formed: an inside-out patch with its cytoplasmic membrane face exposed to the bath solution, and an outside-out patch with its extracellular membrane face exposed to the bath solution. 6. The application of the method for the recording of ionic currents and internal dialysis of small cells is considered. Single channel resolution can be achieved when recording from whole cells, if the cell diameter is small (less than 20 micrometer). 7. The wide range of cell types amenable to giga-seal formation is discussed.

The dependence of onset and duration of sleep on the circadian rhythm of rectal temperature

Abstract: The sleep-wake cycle and the circadian rhythm of rectal temperature were recorded in subjects who lived singly in an isolation unit. In 10 subjects, the freerunning rhythms remained internally synchronized, 10 other subjects showed internal desynchronization. Times of onset and end of bedrest ("sleep") were determined in each cycle and referred to the phase of the temperature rhythm. In the synchronized subjects, onset of sleep occurred, on the average, 1.34 h before the minimum of temperature, and end of sleep 6.94 h thereafter, with narrow distributions. The desynchronized subjects had a broad bimodal distribution of sleep onsets (peaks 6.3 and 1.3 h before the minimum); the duration of sleep varied between more than 15 h when sleep began about 10 h before the temperature minimum, and less than 4 h when sleep began several hours after the minimum. The dependence of sleep duration on body temperature is interpreted as a continuing action of the coupling forces between the two rhythms after mutual synchronization is lost.

Presence of luminal K+, a prerequisite for active NaCl transport in the cortical thick ascending limb of Henle's loop of rabbit kidney.

Abstract: Previous data from our laboratory have shown that active transport in the cortical thick ascending limb of Henle's loop (cTAL), as measured by the short circuit current (Lsc, microA X cm-2), requires the presence of Na+ and Cl-. The data were compatible with the model of secondarily active Cl- reabsorption involving the cotransport of Na+ and Cl- across the luminal membrane. The data suggested, furthermore, that 1 Na+ and 2 Cl- interact with the luminal carrier. In the present study it was tested whether this reabsorptive mechanism also requires the presence of luminal K+. Isolated cTAL segments (n = 40) were perfused at high flow rates with a modified Ringer's solution. Removal of K+ from the lumen reduced Isc significantly from 215 to 133 microA X cm-2. Addition of Ba2+ (10(-3) mol X 1(-1)) which blocks the K+ conductance of the luminal, membrane, to the K+-containing lumen perfusate decreased Isc significantly from 234 to 141 microA X cm-2. Combination of both manoeuvres: perfusion with a K+-free and Ba2+-containing solution almost abolished Isc from a control of 237 to 56 microA X cm-2. The results are compatible with the view that in rabbit cTAL the luminal carrier interacts with all 3 ions, possibly 1 Na+, 2 Cl-, and 1 K+. K+ recycles across the luminal membrane through its conductive pathway.

Cation selectivity of the isolated perfused cortical thick ascending limb of Henle's loop of rabbit kidney.

Abstract: The cation permeability of the cortical thick ascending limb of Henle's loop was investigated with electrophysiological methods in the isolated perfused tubule preparation of rabbit kidney. The transepithelial specific resistance (R T) and electrical potential difference (PD) were determined in 4 experimental groups. In group 1 (n=51) the tubules were perfused with a modified Ringer's solution on both sides of the epithelium; the PD was 7±0.4 mV lumen positive, and theR T 34±2 Ω·cm2. In group 2 (n=12) one of both sides of the epithelium was perfused with dilute (54 mmol/l) NaCl solutions under control conditions and in the absence of active transport. Inhibition was obtained in four different ways: low temperature (22° C), zero K+ solutions on both sides of the epithelium, 5·10−5 mol/l furosemide, added to lumen perfusate, or 10−5 mol/l ouabain added to the bathing solution. In the presence and in the absence of active transport a NaCl gradient of 154 versus 54 mmol/l induced diffusion potentials across the epithelium which were symmetrical and of nearly equal magnitude: +12, −14 and +15, −14 mV respectively. In group 3 (n=51) Na+ was completely replaced by choline+, tetraethylammonium+, tris-hydroxymethyl-aminomethane+, or Li+ in either bath or lumen perfusate or in both perfusates. The biionic diffusion potentials were symmetric; the replacement of Na+ by these cations on both sides markedly increasedR T. Both kinds of measurements yielded a permeability sequence ofP Na +>P Li +>P organic cation. In group 4 (n=17) 50 mmol/l of Na+ was replaced by K+, Li+, Rb+, or Cs+ on one of the sides and active transport was inhibited by furosemide or ouabain. From the membrane diffusion potentials and theR T values in group 4 as well as in group 3 the following cation permeability sequence was calculatedP K +>P Na +>P Rb + =P Li +>P Cs +>P organic cation. It is concluded that the cortical thick ascending limb of Henle's loop has a low resistance pathway which is cation selective similar to that of “leaky” epithelia. Since the membrane diffusion potentials are symmetric and since they are not altered by inhibition of active transport, it is likely that this low resistance pathway is formed by a paracellular shunt.

Muscle weakness following eccentric work in man.

Abstract: The separate effects of negative (eccentric) and positive (concentric) work on stimulated and voluntary isometric contraction of the triceps surae were studied in five healthy male subjects following the performance of boxstepping for 1 h with a constant leg lead. The results showed unequivocally that the long-lasting muscle weakness which arises from box-stepping was due to the negative component of the work. The maximal twitch ( $$P_{ t_{_0 } } $$ ) and tetanic (P 0) tensions at 10, 20, 50 and 100 Hz were markedly reduced in the leading (“negative”) leg, which was constantly required to absorb the force of body weight as the subject returned from box to floor, whereas the trailing (“positive”) leg which lifted body weight from floor to box was relatively unaffected. The decreases in $$P_{ t_{_0 } } $$ andP 0 at 20 Hz in the “negative” leg following work were 38 N and 765 N (25% and 55%) respectively and recovery was slow (>20 h). A 2 min “fatigue” test reflected the changes inP 0; a reduction of absolute force was only seen in the negative leg though the relative (%) decline in tension was the same in both legs and its rate did not differ significantly following exercise from control conditions, which suggests the muscles were weaker but not more fatiguable following exercise. At submaximal voltages of stimulation muscle tensions at 20 and 50 Hz were enhanced in the “positive” leg but depressed in the “negative” leg, the relative (but not absolute) loss of force being greater at 20 Hz than 50 Hz which gave rise to long-lasting fall in the 20/50 tension ratio. The loss of high frequency (50 Hz) force was associated with changes in MVC: MVC (N)=507+0.937P 0 (N);r=+0.846. It was suggested that repeated stretching of the triceps surae muscles during the eccentric phase of work in the “negative” leg could affect the efficacy of the contractile machinery directly and may cause actual muscle damage. The subjects complained of muscle soreness for 5–7 days after the experiments.

Chloride reabsorption in the rabbit cortical thick ascending limb of the loop of Henle. A sodium dependent process.

Abstract: The question was investigated whether the chloride reabsorption in the cortical thick ascending limb (cTAL) of rabbit kidney is primary or secondary active, i.e. whether it depends on sodium. Isolated cTAL segments were perfused in vitro at high flow rates (10–20 nl·min−1) with identical modified Ringer's solutions on both sides of the epithelium. The modified Ringer's solution contained sodium and/or organic cations (tris-hydroxymethyl-aminomethane, tetraethylammonium, choline) and 150 mmol·l−1 chloride. Transepithelial electrical potential difference (PD) and transepithelial specific resistance (R T) were directly measured, and used to calculate the short circuit current (Isc) under three types of experimental conditions. In group 1 (n=8), the tubules were first perfused with solutions containing 150 mmol·l−1 sodium. The PD was +9.6±0.4 mV (relative to the tubule lumen). Then sodium was replaced by an organic cation whereby the PD fell slightly to +8.2±1.6 mV. This PD disappeared when furosemide was added to the lumen perfusate, ouabain added to the bath, or temperature decreased to 295°K (22° C). To test whether the removal of sodium was successfull in this series, the sequence of perfusions was reversed in the second group (n=31). First the perfusion system was thoroughly rinsed with a sodium-free solution. Then a tubule was mounted and perfused under seemingly sodium-free conditions. The PD was only + 2.6 ±0.3 mV, corresponding to an Isc of 29±3 μA·cm−2. When sodium was gradually added, PD and Isc increased steeply with a half maximal response at 3.4 mmol·l−1. The maximal Isc was 258 μA·cm−2. In the last series (n=13), the efforts to make the system sodium-free were even more rigorous. The perfusion system in this series was made from sodium-free glass and the bath exchange rate was increased. The PD in the absence of sodium was +0.6±0.2 mV. These data indicate that the apparent sodium-independence in series 1 as well as in previous reports on this issue is artefactual and is caused by a small amount of sodium which is left in the system after the replacement by sodium-free solutions. A kinetic analysis of series 2 and 3 revealed that all active chloride reabsorption by the cortical thick ascending limb of Henle's loop is sodium-dependent suggesting that sodium and chloride are cotransported in the luminal membrane of this nephron segment.

The effect of reduced calcium on quantal unit current and release at the crayfish neuromuscular junction.

Abstract: Excitatory postsynaptic currents (EPSCs) were recorded extracellularly from large muscle fibers by means of ‘patch clamp’ electrodes. Compared to usual extracellular recordings, better signal/noise ratio and temporal stability were achieved. In the range of extracellular calcium concentrations [Ca]0 between 2.7 and 13.5 mmol/l (normal), the average amplitude of the EPSC increased more than proportional to [Ca]0. The unit quantum current,C1, and the average release rate,m, were determined from EPSCs and also from spontaneous sEPSCs, using both Poisson and binomial statistics. The main effect of [Ca]0 was onm: at different synaptic sitesm depended on the second to fourth power of [Ca]0. In terms of binomial parameters, the release probabilityp is the [Ca]0-dependent one. In addition, reduction of [Ca]0 from 13.5 to 2.7 mmol/l decreased the unit quantumC1 consistently to 60%; simultaneously the rise and decay of EPSCs and sEPSCs were shortened by 10–20%. [Ca]0 thus has strong presynaptic effects on the release probability, but in addition smaller ones on teh postsynaptic channel characteristics.

Influence of noradrenaline on blood flow to brown adipose tissue in rats exhibiting diet-induced thermogenesis

Abstract: 1. The influence of noradrenaline on regional blood flow was determined using radioactive microspheres in rats maintained on either stock diet or a palatable cafeteria diet. 2. Cardiac output and blood flow to brain, lungs, liver and skeletal muscle were similar for rats on the two diets. 3. Blood flow to total dissectable brown adipose tissue in control and cafeteria rats represented 1 and 2% of cardiac output respectively but these values rose to 7 and 15.5% during infusion of noradrenaline. 4. Arterial oxygen content was similar for all groups but the oxygen content of venous blood draining the interscapular brown adipose tissue fell to 6 ml O2/100 ml blood in control rats and 1 ml/100 ml in cafeteria rats after noradrenaline. 5. The total oxygen consumption of brown adipose tissue was calculated and found to account for 42% of the response to noradrenaline in control rats and 74% in cafeteria animals. The increments in the oxygen consumption of other tissues were almost identical in both groups and so all the diet-induced changes in thermogenic capacity can be attributed to increases in brown adipose tissue metabolism. 6. These findings demonstrate the quantitative importance of brown adipose tissue in diet-induced thermogenesis and confirm the similarities between diet and non-shivering thermogenesis.

Influence of osmotic compression on calcium activation and tension in skinned muscle fibers of the rabbit

Abstract: Single skinned muscle fibers were osmotically compressed back to and below their in situ size by addition of a large, random-coil polymer (Deytran T500; $$\bar M_{\text{N}}$$ = 180,000; $$\bar M_{\text{W}}$$ = 461,000) to the bathing medium. Maximal Ca2+-activated tension in fibers swollen (zero Dextran, fiber width 21% above in situ) or near in situ size (5% Dextran, in g/100 ml final solution) was similar, but compression to 86% of in situ width with 10% Dextran decreased maximal force by 15% relative to polymer-free control. While the relative tension-pCa relation in 0 and 10% Dextran was similar, with a pCa of 6.37 required for 50% activation, that in 5% Dextran was more sensitive to Ca2+, with a pCa50 of 6.66. We feel these effects are most likely due to changes in interfilament spacing with compression and that alterations in Ca2+-sensitivity might be explained by changes in cross-bridge angle or in the concomitant attachment-detachment rate constants which would be expected to influence the troponin-Ca2+ binding equilibrium, as has been proposed by others.

Heat stress effects on capillary blood flow and its redistribution in the laying hen.

Abstract: The effect of heat stress on capillary blood flow (CBF) distribution was examined in laying hens, using 15 micron microspheres, by determining CBF before and after elevating body temperature by 1–2°C. No change was evident in unfeathered metatarsal skin, although its temperature increased by 7°C. Breast skin CBF change was 3.5 times larger than that of back skin. Comb CBF increase was larger than in wattles. CBF in upper respiratory tract increased proportionally to increment in respiratory frequency. Digestive system CBF was reduced by hyperthermia: the effect was pronounced in its upper organs (46% of normal) and decreased along the tract. CBF increased 4-fold in an expiratory abdominal muscle, a smaller rise occurred in a pectoral muscle and no change in a leg muscle. CBF in the tibia fell to 64% of normal. In the reproductive system, CBF fell to 58% of control level in the uterus, to 70–80% in the larger ovarian follicles and infundibulum with no significant changes in magnum and isthmus. Cerebral CBF increased during hyperthermia.

Single channel activity associated with the calcium dependent outward current in Helix pomatia.

Abstract: A recently improved version of the extracellular patch clamp technique (9, 13) was used to record currents from microscopic membrane areas of Helix neurons with predominant Ca2+ dependent outward currents. Current fluctuations in the patches consisted mainly of frequently interrupted, one-sided steps indicating discrete open-closed state changes of single channels with an ohmic conductance of approximately 19 pS. Frequency of occurrence of the elementary events compares with amplitudes of macroscopic currents during depolarizing voltage steps of varied amplitude. Average delays in appearance of the events vary in line with delayed time courses of the cell's outward current.

Current source density analysis: methods and application to simultaneously recorded field potentials of the rabbit's visual cortex.

Abstract: This paper deals with the application of current source density (CSD) analysis to simultaneously recorded intracortical field potentials of the rabbit's visual cortex. Recordings were made with multielectrodes with either 8 contacts at distances of 300 microns, or 16 contacts at distances of 150 microns on one carrier needle. For synchronized activities, a spatial resolution of 150 microns turned out to be sufficient to record all depth-varying details of the field potentials; for seizure potentials even a spacing of 300 microns was adequate in most cases. For practical application, an appropriate spacing of the measuring points has to be chosen for a satisfactory estimation of the first and second derivatives of the field potentials. For this reason an interpolation procedure is applied to reduce the spacing from 300 microns or 150 microns electrode contact distances, respectively, and to obtain intermediate values at 75 microns distances. With this spacing satisfactory estimations of the second derivative are obtained. Theoretically, CSD analysis has to be made three-dimensionally, but under certain conditions which are discussed, a one-dimensional analysis can be applied. An unknown quantity is sigma z, the vertical conductivity. It turned out that average values obtained from different experiments are not representative and that the vertical conductivity has to be measured in every experiment. This is caused by the great individual differences of the cortices even if the same stereotactic coordinates are chosen. Therefore, in every experiment relative conductivity measurements are performed. The influence of different conductivity values within the various layers and the influence of a conductivity gradient is discussed and demonstrated by examples.

The early phase of experimental acute renal failure. VI. The influence of furosemide.

Abstract: Tubular obstruction in acute renal failure, postulated to cause the restricted excretory function, is suggested by raising intratubular pressure, to lower effective filtration pressure and diminish urine output. To examine the applicability of the obstruction hypothesis to the pathogenesis of experimental acute renal failure, proximal intratubular pressure and renal function were measured after renal insults of different origins and severity. Obstruction in acute renal failure kidneys should manifest itself as an increase in intratubular pressure for a least 12 h, for within this time period following ureteral occlusion, elevated pressures were found to reflect obstruction. The consistent existence of raised proximal intratubular pressure in acute renal failure kidneys could not be detected; ischaemic and nephrotoxic models were found in which no rise in intratubular pressure could be demonstrated. The oliguric nature of acute renal failure kidneys could not be verified; ischaemic and nephrotoxic models were found in which urine output was either normal or enhanced. Only for methaemoglobin induced renal failure were raised intratubular pressure, oliguria and casts concurrent. It is concluded that obstruction is not a consistent feature of experimental acute renal failure and that the obstruction hypothesis may be specifically applicable to only a few models, which include haeme pigment and folic acid induced renal failure.

A Modified System for in vitro Perfusion of Isolated Renal Tubules

Abstract: A modified system for the in vitro perfusion of isolated tubule segments is presented. The system consists of four holders each of which carries an acrylic cylinder. The acrylic cylinders are used to fix the glass pipettes in a concentric position. The four holders are mounted onto a support consisting of two holding pieces and three steel rods. Three of the holders contain ball-races so that they can slide on the rods with high accuracy and little friction. The holers to which the sylgard pipette and the perfusion pipette are attached are moved by electric motors. Compared with the classical V-track system this modification has the advantage of higher precision. Once the different pipettes are centered, concentricity is maintained even when the pipettes are moved forward or backward. Thus, this equipment facilitates the cannulation of tubules and increases the number of successfull experiments.

The time courses of intracellular free calcium and related electrical effects after injection of CaCl2 into neurons of the snail,Helix pomatia

Abstract: Controlled quantities of 100 mM aqueous CaCl2 solutions were pressure injected into voltage-clamped neurons with a resolution of 10−11 1. Ca2+-selective microelectrodes monitored the time course of changes in [Ca2+]i. At a membrane potential of −50 mV CaCl2 quantities in the range of 1% of the cell volume induced an inward current, associated with a conductance increase and having an equilibrium potential between −20 and +20 mV, which accompanied the rise in [Ca2+]i. An artifactual origin of the inward current by the injection procedure or by calcium screening of membrane sites could be excluded. The calcium-induced hyperpolarizing conductance, producing an outward current at −50 mV, followed the inward current and reached maximum during the late decline in [Ca2+]i. In most cases its development was separated from the inward current by an intermediate relative decrease of the membrane conductance. Neither of the two transient conductance increases showed a particular dependence on voltage. Renewed Ca2+ injection quickly decreased the calcium-induced hyperpolarizing conductance for several seconds. Ca2+ injections below 0.05% of the cell volume mostly produced pure outward currents or hyperpolarizing responses. Partial substitution of extracellular CaCl2 by NiCl2 decreased the hyperpolarizing response but not the initial inward current. The immediate effects of increased [Ca2+]i are activation of a depolarizing conductance and the partial block of the late hyperpolarizing conductance. The latter is probably produced through intermediate steps after increasing [Ca2+ i.

Influence of blood flow on fatty acid mobilization form lipolytically active adipose tissue.

Abstract: Subcutaneous, ingvinal adipose tissue from dogs was perfused with blood to which had been added isoprenaline and theophyllamine in order to stimulate lipolysis. The supply of free fatty acid (FFA) carrier to the tissue was varied either by variations in the rate of blood flow or by changes in the albumin concentration of the perfusing blood at constant flow rate. The net production of FFA from the tissue was found to depend on the supply of carrier over a range from 0.1–12 μmoles of albumin × 100g tissue−1 × min−1. The corresponding molar\(\frac{{FFA}}{{albumin}}\) ratios in adipose venous blood varied between 12.1 and 1.2. The changes in FFA production appeared to be due to varying degrees of reesterification rather than changes in the rate of lipolysis. The findings suggest that the increase in adipose tissue blood flow demonstrated during various lipolytic conditions is of physiological importance by facilitating the removal of FFA from adipose tissue. Equilibration experiments showed the FFA binding capacity of both dog serum and human plasma to be above that calculated from the association constants of purified human albumin, suggesting the binding of FFA to other plasma carriers than albumin.

Functional disconnection of brown adipose tissue in hypothalamic obesity in rats

Abstract: The metabolic responses to electrical nerve stimulation, norepinephrine or octanoate additions were studied using continuous monitoring of NAD(P)H/NADP redox state by reflexion spectrophotometry of interscapular brown adipose tissues from control and ventromedial hypothalamic (VMH) lesioned rats. The responses to these stimuli were all greatly decreased already 3 days after VMH lesions, indicating a reduced cell capacity to oxidize free fatty acids. Measurements of interscapular brown adipose tissue composition 4–5 weeks after VMH lesions showed a decrease of both DNA concentration and total content, indicating some tissue involution.

Lognormal distribution of intercapillary distance in normal and hypertrophic rat heart as estimated by the method of concentric circles: its effect on tissue oxygenation

Abstract: The inhomogeneity of the capillary net in the cardiac muscle was estimated using our morphometric measurements in normal and hypertrophic rats hearts. As entry data we used the distribution of tissue at different distances from the nearest capillary as measured by the method of concentric circles and the mean intercapillary distance independently calculated from the capillary density. The derived distribution of intercapillary distances was approximated by lognormal distribution in which the spread can be characterized by a single parameter, namely the log standard deviation. The effect of the log standard deviation on tissue oxygenation was evaluated in normal and hypertrophic hearts, at normoxia and at hypoxia. The mean tissueP O2 and the percentage of anoxic tissue at the venous end of the tissue cylinder were calculated using Krogh's model. Two boundary situations were considered: A) the end-capillaryP O2 was assumed to be equal in all capillaries due to compensatory adjustment in blood flow; B) the same flow in all capillaries was assumed resulting in varying end-capillaryP O2. The real situation is expected to be between situations A and B. Increased variability of intercapillary distance proved to impair considerably the tissue oxygenation, especially when the results were expressed as a percentage of anoxic tissue. The percentage of anoxic tissue turned out to be a better index of tissue oxygenation than the meanP O2 particularly at hypoxia. The results suggest the presence of at least a partial adjustment of blood flow with respect to the width of tissue cylinder. Without such adjustment, a large part of tissue would become anoxic already in normal hearts at normoxia and this would be further aggravated by hypertrophy and/or hypoxia.

Effects of parathyroid hormone and N6,O2'-dibutyryl cyclic AMP on Ca2+ transport across the rabbit distal nephron segments perfused in vitro.

Abstract: Effects on Ca2+ transport of parathyroid hormone (PTH) and N6,O2'-dibutyryl adenosine 3',5'-cyclic monophosphate (DB-cAMP) were examined in the rabbit distal nephron segments including the cortical thick ascending limb of Henle's loop (CAL), the connecting tubule (CNT) and the cortical collecting tubule (CCT) by the in vitro perfusion technique. When PTH (10(-8) mol . l-1) was added to the bath, efflux of Ca2+ (pmol . mm-1 . min-1) was increased from 6.29 +/- 1.46 to 7.96 +/- 1.66 (P less than 0.02) in the CAL, and from 8.55 +/- 1.30 to 13.73 +/- 1.24 (P less than 0.001) in the CNT, respectively, without changes in influx of Ca2+. The effect of PTH on Ca2+ transport in the CAL, however, was abolished when phosphate concentration in the medium was reduced from 3.0 to 1.0 mmol . l-1. When DB-cAMP (10(-3) mol . l-1) was added to the bath, efflux of Ca2+ was also increased from 7.01 +/- 0.83 to 9.40 +/- 0.82 (P less than 0.05) in the CAL, and from 13.11 +/- 0.89 to 19.74 +/- 0.52 (P less than 0.005) in the CNT, respectively. By contrast, neither PTH nor DB-cAMP affected efflux of Ca2+ in the CCT. PTH did not affected the transepithelial voltage either in the CAL or in the CNT. But in the CNT, DB-cAMP decreased the voltage from -14.1 to -9.4 mV. The response of adenylate cyclase activity to PTH in the collagenase treated isolated nephron segments was also examined. Significant increases in adenylase cyclase activity were observed in the CAL as well as in the CNT with 10(-6) mol . l-1 PTH. These data indicate that PTH stimulates Ca2+ transport across the CNT probably via activation of the adenylate cyclase-cyclic AMP system. The hormone may also stimulate Ca2+ transport across the CAL in a special condition where plasma phosphate concentration is elevated.

Bicarbonate reabsorption in the papillary collecting duct of rats.

Abstract: Using the technique of capillary perfusion and simultaneous luminal stop flow microperfusion the reabsorption of bicarbonate and glycodiazine from the papillary collecting duct was evaluated. Starting with equal H14CO3− and3H-glycodiazine concentrations in the luminal and peritubular perfusates, the decrease in the luminal concentration at 10 and 45 s contact time was measured.

A study of the amygdaloid defence reaction showing the value of Althesin anaesthesia in studies of the functions of the fore-brain in cats.

Abstract: 1. In cats under Althesin (alphaxalone-alphadalone) anaesthesia, sites in the amygdala and brain-stem defence areas have been electrically stimulated by means of monopolar, semi-microelectrodes. 2. Such stimulation evoked a consistent pattern of visceral changes characteristic of the alerting stage of the defence reaction as it has been described by previous workers. This “visceral alerting reaction” included increases in arterial blood pressure, heart-rate and cardiac output with vasoconstriction in kidney, intestines and skin but vasodilatation in the hind limbs. 3. These results differ strikingly from those reported previously in that, under conventional anaesthetics, such as chloralose or barbiturate, the full visceral alerting reaction cannot be evoked by amygdala stimulation, or any other manoeuvre which involves transmission through the brain stem defence areas. 4. The area of the amygdala from which such responses can be elicited under Althesin closely resembles that which has been reported to evoke defence reactions in conscious animals. 5. It is concluded that Althesin, used in the manner described, does not distort synaptic transmission in the forebrain in the way that conventional anaesthetics do. It is suggested that this steroid anaesthetic may be invaluable in any studies of fore-brain physiology in the cat

Effect of hyperthyroidism on the contractile and histochemical properties of fast and slow twitch skeletal muscle in the rat.

Abstract: Male Wistar rats were made hyperthyroid by intraperitoneal injections on alternate days for 1–6 weeks, of 200 μg/kg triiodothyronine (T3). The effects of this treatment on the contractile properties of the soleus, a slow twitch and the extensor digitorum longus (EDL), a fast twitch skeletal muscle, were studied in vivo in the anaesthetized animal. Post mortem, serial frozen sections of both muscles were stained histochemically for myosin ATPase, succinic dehydrogenase and phosphorylase. Muscle fibres were classified as either slow twitch (SO), fast twitch oxidative glycolytic (FOG) or fast twitch glycolytic (FG).

The influence of pH on phosphate transport into rat renal brush border membrane vesicles.

Abstract: Sodium-dependent transport of inorganic phosphate into brush border membrane vesicles is strongly influenced by altering pH of the incubation medium (pHo). At constant total phosphate concentration an increase in pHo leads to an increase in the uptake of inorganic phosphate. Uptake of inorganic phosphate, however, is not affected by the intravesicular pH (pHi) or by transmembrane pH differences (pHo-pHi). If initial phosphate uptake is studied as a function of total phosphate concentration in the medium the half saturation concentration increases when pHo is raised from 6.3–6.9 but remains unaltered between pHo 6.9 and 7.8V max increases about 3-fold between pHo 6.3 and 6.9 and by a factor of about 1.6 between pHo 6.9 and 7.4. The pHo-dependence of phosphate uptake is diminished by increasing sodium concentrations. Altering transmembrane electrical potential difference by potassium + valinomycin-induced diffusion potentials or by anion replacement fails to demonstrate electrogenicity of sodium-phosphate cotransport. Experiments using a potential-sensitive fluorescent dye, however, indicate a vesicle inside positive electrical potential difference when inorganic phosphate is added. The phosphate-induced alterations in the electrical potential difference are sodium-dependent and more pronounced at low pHo values. Together with earlier observations there results suggest that translocation of inorganic phosphate across the proximal tubular brush border membrane is mediated by cotransport of 2 sodium ions with one either monovalent or divalent phosphate molecule according to its availability in the tubular fluid. The pH sensitivity of this transport system is rather due to alterations in the transport system itself than to pH-dependent alteration in the ratio of monovalent to divalent phosphate.

Rapid rise in plasma glucagon induced by acute cold exposure in man and rat.

Abstract: The effect of acute cold exposure on the concentration of glucagon in the blood was investigated in man and in intact and adrenalectomized rats. In man fasted overnight acute cold exposure, which caused a twofold increase in O2-consumption resulted in a rapid rise in plasma glucagon. The levels of insulin and blood glucose remained unaltered, while the concentration of serum free fatty acids and β-hydroxybutyrate increased. In fasted intact rats acute cold exposure lead to similar effects. A close parallelism between the rise in plasma glucagon and the concentration of hepatic cycloAMP was observed. Adrenalectomy did not impair the cold induced rise in plasma glucagon and hepatic cycloAMP. It is concluded that acute cold exposure caused a rapid rise in the concentration of plasma glucagon leading to an increase in the concentration of hepatic cycloAMP, thus enhancing the rate of hepatic gluconeogenesis and ketogenesis. As these alterations were similar in the absence of glucocorticoids and medulla-derived catecholamines, it is suggested that glucagon may play a role in the metabolic adaptation to acute cold exposure.

Electromyographic fatigue analysis based on the number of zero crossings.

Abstract: An alternative method for analysis of localized muscle fatigue as performed by Lindstrom et al. (1977) is presented. The method is based on counting of zero crossing of the electromyographic signal. The method is implemented on a microprocessor computer system. The analysis can be done on line and the result is presented as a plot of the normalized action potential velocity versus time.

Sodium-chloride transport in the thick ascending limb of Henle's loop. Oxygen consumption studies in isolated cells

Abstract: Isolated cells were prepared from the medullary thick ascending limb of Henle's loop (TALH) and the response of oxygen consumption was correlated with the active chloride transport system found in these cells. Oxygen consumption was 31.6 μl O2/mg protein·h and inhibited 50% by the absence of either sodium or chloride in the incubation medium. The absence of both sodium and chloride produced no further inhibition of oxygen consumption. Ouabain (10−4 M) inhibited oxygen consumption by 50% and the inhibitory effect depended on the presence of both sodium and chloride in the incubation medium. Further, furosemide inhibited oxygen consumption by a maximum of 50% at 10−3 M and also had no inhibitory effect if either sodium or chloride were absent. Furosemide had no effect on the Na, K-ATPase activity or ATP levels of the TALH cells. Thus, the data suggest that 50% of the oxygen consumption of the TALH cells is related to the movement of sodium and chloride into the cell and that the ions may be transported in a coupled manner.

Tension response and 45Ca release in vascular smooth muscle incubated in Ca-free solution.

Abstract: The contractile response of arterial smooth muscles induced by agonists as noradrenaline or histamine in Ca-free solution consists of two phases: an initial phasic component, which is transient and accompanied by a release of cellular Ca, and a small tonic component, which persists as long as the agonist is present. A second admission of the agonist without reexposure to Ca elicits only the tonic component. This tonic contraction differs in several respects from the phasic response obtained in Ca-free solution: it is independent of the duration of exposure to the Ca-free solution, it can be elicited many times without reexposure to Ca, and it is not accompanied by a measurable release of Ca from the cells. During superfusion with Ca-free solution, a tonic contraction is also induced by fluoride ions at concentrations exceeding 4 mM. The amplitude of this contraction is maximal at about 12 mM. Increasing the fluoride concentration shortens the delay between the addition of the F− and the onset of the contraction. As is the case for the tonic noradrenaline-response, the F−-induced contractions can be clicited many times without reexposure to Ca. The tonic contractions evoked by noradrenaline or histamine and by fluoride ions are additive. Both contractions are reversibly inhibited by caffeine, theophylline, Na-nitroprusside, papaverine and by nitroglycerine. The possiblity that these tonic contractions are not accompanied by an increase of the cytoplasmic Ca2+ concentration is discussed.

Block of Na channels in the membrane of myelinated nerve by benzocaine

Abstract: 1. The actions of the neutral local anesthetic benzocaine on Na channels were studied in voltage-clamp experiments on single myelinated nerve fibres of the frog by measurement of sodium currents, asymmetry currents, and sodium current fluctuations. 2. 1 mM benzocaine reduced the peak Na currents during various depolarizationsV between 20 and 120 mV to 63% of their control values but did not change the time constant of Na activation. 3. 1 mM benzocaine altered the asymmetry currents during 1 ms pulsesV between 20 and 120 mV in the same way as the early Na currents: It reduced the amplitude to 64% but did not affect the kinetics of the currents. 4. The charge displacement of the asymmetry current during the pulse (Qon) was compared with the charge displacement after the pulse (Qoff). Without benzocaine the relative chargeQoff/Qon declined to a constant level (0.42 atV=40 mV, 0.25 atV=100 mV) with increasing pulse durations. In the presence of 1 mM benzocaine the chargesQoff after pulses toV=40 or 100 mV are almost independent of pulse duration and approximately equal to the controlQoff values after 5.6 ms pulses. Thus, the immobilizations caused by Na inactivation and benzocaine are not additive. 5. Na currents and Na-current fluctuations were recorded during depolarizationsV between 24 and 48 mV in the presence of 0.1 mM benzocaine and 7 μMAnemonia toxin II. A lower limit of 8.6 pS was derived for the conductance of a single Na channel. The value agrees with other estimates of the conductance of Na channels which had not been treated by local anesthetics. This suggests an “all-or-none blocking” of Na channels by benzocaine.

Comparative rheology of nucleated and non-nucleated red blood cells. I. Microrheology of avian erythrocytes during capillary flow.

Abstract: Deformation and dynamic flow behaviour of nucleated duck erythrocytes were compared to those of nonnucleated human cells during steady flow (at wall shear stresses up to 3 N.m-2) through glass capillaries (diameter between 5 micrometer and 12 micrometer). While the deformation process of both cell species is qualitatively similar, significant quantitative differences demonstrate a lower deformability of the nucleated erythrocytes. In comparison to human cells, the avian red cells exhibit significantly lower stability of orientation during capillary flow. Viscometric measurements show that, in contrast to human red cell suspensions, the relative viscosity of nucleated cell suspensions does not decrease with increasing suspending medium viscosity. The results indicate that the hydrodynamic disturbance caused by avian erythrocytes in the flow of the suspending fluid is more pronounced than that of the mammalian cells. This is due to their lower ability to adapt to local shear forces.

Single channel Ca2+ currents in Helix pomatia neurons.

Abstract: Unitary Ca2+ currents of TEA injected Helix neurons were recorded in the “Giga seal” situation (6, 7) from microscopic membrane patches exposed to 50 mM [Ca2+]o, O [Na+]o, 20 mM [TEA+]o and 2.5 μM [TTX]o. Constant field assumptions yield a channel permeability of 2.9±1.0×10−14 cm3s−1 corresponding to slope conductances of 5 to 15 pS between 0 and −30 mV. Frequency of occurrence of the units strongly increased with depolarization. Mean open time of the Ca2+ channels was about 3 ms without obvious dependence on voltage. A similar open time was seen with [Ba2+]o, yielding about double the current strength when compared with [Ca2+]o.