The Kinetics of Thermal Injury in Human Renal Carcinoma Cells

In this study, the thermal injury behavior of both suspended and attached SN12 human renal carcinoma cells (RCC) under thermal therapy conditions (i.e., heating cells to elevated temperature for seconds to minutes) was investigated using a non-isothermal method. This non-isothermal method entailed heating the cells using a programmable heating stage from room temperature at 130C min^–1 to various peak temperatures from 45 to 70C, held for 0–10 min, and then cooling down to room temperature at 65C min^–1. It was found that the suspended SN12 cells are more heat susceptible than attached ones. The non-isothermal portions (i.e., the heat-up and cool-down portions) of the thermal histories were found to be able to cause significant injury (> 10%) in both suspended and attached SN12 cells when the peak temperature is above 60C. Therefore, a non-isothermal method, which accounts for both the isothermal and non-isothermal portions of the thermal histories, was used to extract the kinetic parameters (i.e., the activation energy and frequency factor) in the Arrhenius injury model for SN12 cells. Furthermore, these results suggest that this non-isothermal method can be used to extract kinetic parameters from in vivo heating studies using minimally invasive surgical probes, where it is very difficult to get a thermal history in tissue with a dominant isothermal portion.     

Control of single-joint movements in deafferented patients: evidence for amplitude coding rather than position control

Two deafferented patients and several control subjects participated in a series of experiments to investigate how accurate single-joint movements are programed, spatially calibrated, and updated in the absence of proprioceptive information. The deafferented patients suffered from a permanent and severe loss of large sensory myelinated fibers below the neck. Subjects performed, with and without vision, sequences of forearm supinations and pronations with two temporal delays between each movement (0 s and 8 s). Overall, the lack of proprioception did not yield any significant decrease in movement accuracy when vision was available. Without vision, the absence of proprioceptive afferents yielded (1) significantly larger spatial errors, (2) amplitude errors similar to those of control subjects, and (3) a significant drift when an 8-s delay was introduced between two successive movements. Subjects also performed, without vision, a 20 supination followed by a 20 pronation that brought back the wrist to the starting position. On some trials, the supination was blocked unexpectedly by way of a magnetic brake. When the supination was blocked, subjects were already on the second target and no pronation was required when the brake was released. The defferented patients, unaware of the procedure, always produced a 20 pronation. These data confirm that deafferented patients were not coding a final position. It rather suggests that they coded an amplitude and translated the spatial distance between the two targets in a corresponding force pulse. Overall, the results highlight the powerful and key role of proprioceptive afferents for calibrating the spatial motor frame of reference.     

Measured and modeled properties of mammalian skeletal muscle. I. The effects of post-activation potentiation on the time course and velocity dependencies of force production

Activation of mammalian fast-twitch skeletal muscle induces a persistent effect known as post-activation potentiation (PAP), classically defined as an increase in force production at sub-maximal levels of activation. The underlying mechanism is thought to be phosphorylation of the myosin regulatory light chain (MRLC), which leads to an increase in the rate constant for cross-bridge attachment (Sweeney et al., 1993). If true, this suggests the hypothesis that other contractile properties should be affected during PAP. Using a feline fast-twitch whole-muscle preparation (caudofemoralis) at 37C, we observed that PAP greatly increased tetanic forces during active lengthening, decreased isometric tetanic rise times and delayed isometric tetanic force relaxation. The first two of these effects were length dependent with a greater effect occurring at shorter lengths. These findings confirmed that PAP has other functionally important effects beyond a simple increase in sub-maximal isometric forces. Furthermore, length was found to have an effect independent of PAP on the shortening half of the FV relationship (less force was produced at longer lengths) and on the rate of force relaxation during the later stages of isometric tetanic force decay (slower relaxation at longer lengths). All of these findings can be explained with a simplified, two-state model of cross-bridge dynamics that accounts for the interaction of both interfilament spacing and MRLC phosphorylation on the apparent rate constants for cross-bridge attachment and detachment. These findings are largely consistent with data collected previously from reduced preparations such as skinned fibers at cold, unphysiological temperatures (e.g. 5C). One finding that could not be explained by our model was that twitch fall times in the dispotentiated state were parabolically correlated with length, whereas in the potentiated state the relationship was linear. The time course of decay of this effect did not follow the time course of force dispotentiation, suggesting that there are other activation-dependent processes occurring in parallel with MRLC phosphorylation.     

Activation of atrial muscarinic K+ channels by low concentrations ofβγ subunits of rat brain G protein

Effects of G protein subunits from rat brain on cardiac K⁺ channel was examined in single atrial cells of guinea-pig, using patch clamp techniques. We found that 10 pM concentration of rat brain subunits preparation could activate the atrial muscarine receptor-gated K⁺ channel (I_K.ACh). Neither the detergent, CHAPS, used to suspend nor the boiled preparation activated I_K.ACh. Furthermore, preincubation of subunits preparation in Mg²⁺-free solution, which easily inactivated -GTP-S, did not affect -activation of I_K.ACh. We concluded, therefore, that subunits themselves can activate I_K.ACh.Supported by the grants from the Ministry of Education, Culture and Science of Japan and from the Calcium Signal Workshop on Cardiovascular Systems     

Phénomènes synaptiques du nystagmus

Summary In a previous work (Horcholle and Ty-Dumont 1968), sets of nystagmic interneurones have been described in the region of the abducens nucleus of the cat. One set — the inhibitory nystagmic interneurones (INI) —is triggered by nystagmogenic stimulation and its firing during nystagmus is in opposite phase to the motor nystagmic discharges.In this study, intracellular recordings from ocular motoneurones and from INIs have been obtained during nystagmus to analyse the synaptic mechanisms of the periodic activity.Motoneurones are identified by antidromic invasion following single shock stimulation of the abducens nerve. Their intracellular record during nystagmus has shown a periodic depolarization of the membrane which is synchronous with the motor nystagmic discharges. Between the phases of depolarization, the motoneurone is not excitable by orthodromic inputs of vestibular origin although it can be triggered by antidromic invasion. A reduction in amplitude of the evoked EPSPs, which is synchronous with the silent period of the motoneurones, could suggest a presynaptic inhibitory mechanism.INIs are identified by their rhythmic firing during nystagmus which is in opposite phase to the nystagmic discharges recorded simultaneously from the abducens nerve. Intracellular records from INIs have shown a periodic hyperpolarization of their membrane which is synchronous with the nystagmic motor discharges. INIs phase of activity coincides with the silent period of the abducens nerve and is synchronous with the inhibitory phase of the inputs of vestibular origin.A functional relation between the motoneurones and the INIs is highly probable and is considered to play a role in the elaboration of the rhythmic activity of nystagmus.

Ce travail a été exécuté avec l'aide de la DRME (contrat n 233/68), du CNRS (contrat n LA.38), de l'INSERM (contrat n 66.014) et de la DGRST (contrat n 66.00.431).     

Measured and modeled properties of mammalian skeletal muscle: III. the effects of stimulus frequency on stretch-induced force enhancement and shortening-induced force depression

Stretch-induced force enhancement and shortening-induced force depression were examined in fast-twitch feline caudofemoralis muscle at 37C. These phenomena were induced by applying ramp length changes during the first 100–200 ms of an otherwise isometric contraction. The effects of various stimulus frequencies ranging from 30 to 120 pps were investigated over lengths ranging from 0.85 to 1.15 L ₀. Distributed asynchronous stimulation of bundles of ventral roots was employed to produce smooth contractions at sub-tetanic stimulus frequencies in whole muscle. Of the two components of force enhancement identified by Noble (1992) we observed only the transient component that decays with time; we did not observe residual force enhancement. The force depression that we observed was symmetrical in almost all respects to the transient force enhancement, and was unlike the shortening-induced de-activation and residual force depression identified by Edman (Edman, 1975; Edman et al., 1993). Both transient force enhancement and depression were independent of work, load and activation. Reversals in the direction of ramp length changes following either an initial stretch or initial shortening were shown to cancel the effects of both transient force enhancement and transient force depression. The distances over which these cancellations could be achieved were different for the lengthening and shortening effects. This asymmetry can be reconciled with the predictions of Huxley's original cross-bridge mechanism by incorporating the recent suggestion that myosin heads can interact with multiple actin binding sites during a single working stroke. We conclude that the types of force enhancement/depression that are most likely to be encountered under physiological conditions are the transient effects observed here, but that even these will have relatively little effect on force production during most natural behaviors.     

Biochemical investigation with regard to infection and immunity of Eimeria acervulina in the fowl

Summary Biochemical investigation of the fluid surrounding the sporocysts in the oocysts (oocyst fluid) showed the presence of the common amino acids, -isoaminobutyric acid, glycerol, an unidentified carbohydrate and proteins.Incubation experiments with D-glucose-¹⁴C (U) revealed the presence of enzymes able to convert glucose into lactic acid and other acids. Inside the sporocysts the common amino acids, glycerol and the unidentified carbohydrate were also present, but -isoaminobutyric acid did not occur. The carbohydrate binding protein carboglutelin, carbohydrate phosphate and small amounts of glucose and fructose were mainly found inside the sporocysts.Incubation experiments of oocyst fluid mixed with sporocysts with D-glucose-C¹⁴ (U) demonstrated an interchange between the fluid and the sporocysts. In the oocyst fluid lactic acid was formed, but inside the sporocysts the conversion appeared to stop mainly with formation of carbohydrate phosphate.Incubation experiments of intestinal pieces both from immunized and not-immunized birds with oocyst fluid, sporocysts and labelled glucose showed that a stronger reaction took place in immunized birds than in not-immunized ones.Similar experiments were performed with not-immunized birds at different days after a primary infection. The reaction of the intestinal wall, which seemed quite normal again since day 19 after infection, was comparable with that observed in immunized birds.Finally the hypothesis is given, that in the first part of the intestine of immunized birds compounds are present originating from the first infection. These compounds might enhance the reaction between the oocyst fluid and the present feed to such an extent that the pH decreases and much epithelial cells are pushed off possibly together with the sporocysts. Then leakage of serum proteins might occur.     

The effect of Ehrlich's ascitic carcinoma on the function of mouse thyroid

Summary The ascitic fluid of Ehrlich's carcinoma contains a factor capable of an antithyroid effect, which is evidenced by a sharp decrease in the I¹³¹ absorption of the thyroid gland. This factor is thermolabile, is cooled with absolute alcohol, and is apparently of a protein or polypeptide nature. As a metabolic product of tumor cells it enters the humoral media of the body and inhibits the thyroid function. The secretion by tumor cells of a substance inhibiting the thyroid function is regarded as a specific manifestation of one of the mechanisms by which the tumor influences a definite system in the body. This substance cannot be attributed to the group of toxohormones because, as distinct from the latter, which has thermal stability, it is a thermolabile compound.(Presented by Active Member of the Academy of Medical Sciences of the USSR V. V. Parin) Translated from Byulleten' Éksperimental'noi Biologii i Meditsiny, Vol. 61, No. 5, pp. 97–100, May, 1966     

On the regulation of the expressed L-type calcium channel by cAMP-dependent phosphorylation

The Ca²⁺ channel subunits _1C-a and _1C-b were stably expressed in Chinese hamster ovary (CHO) and human embryonic kidney (HEK) 293 cells. The peak Ba²⁺ current (I _Ba) of these cells was not affected significantly by internal dialysis with 0.1 mM cAMP-dependent protein kinase inhibitor peptide (mPKI), 25 M cAMP-dependent protein kinase catalytic subunit (PKA), or a combination of 25 M PKA and 1 M okadaic acid. The activity of the _1C-b channel subunit expressed stably in HEK 293 cells was depressed by 1 M H 89 and was not increased by superfusion with 5 M forskolin plus 20 M isobutylmethylxanthine (IBMX). The _1C-a·₂·₂/ complex was transiently expressed in HEK 293 cells; it was inhibited by internal dialysis of the cells with 1 M H 89, but was not affected by internal dialysis with mPKI, PKA or microcystin. Internal dialysis of cells expressing the _1C-a·₂·₂/ channel with 10 M PKA did not induce facilitation after a 150-ms prepulse to +50 mV. The Ca²⁺ current (I _Ca) of cardiac myocytes increased threefold during internal dialysis with 5 M PKA or 25 M microcystin and during external superfusion with 0.1 M isoproterenol or 5 M forskolin plus 50 M IBMX. These results indicate that the L-type Ca²⁺ channel expressed is not modulated by cAMP-dependent phosphorylation to the same extent as in native cardiac myocytes.     

Glucose kinetics during prolonged exercise in euglycaemic and hyperglycaemic subjects

To determine the limits to oxidation of exogenous glucose by skeletal muscle, the effects of euglycaemia (plasma glucose 5 mM, ET) and hyperglycaemia (plasma glucose 10 mM, HT) on fuel substrate kinetics were evaluated in 12 trained subjects cycling at 70% of maximal oxygen uptake (VO_{2, max}) for 2 h. During exercise, subjects ingested water labelled with traces of U-¹⁴C-glucose so that the rates of plasma glucose oxidation (R _ox) could be determined from plasma ¹⁴C-glucose and expired ¹⁴CO₂ radioactivities, and respiratory gas exchange. Simultaneously, 2-³H-glucose was infused at a constant rate to estimate rates of endogenous glucose turnover (R _a), while unlabelled glucose (25% dextrose) was infused to maintain plasma glucose concentration at either 5 or 10 mM. During ET, endogenous liver glucose R _a (total R _a minus the rate of infusion) declined from 22.4±4.9 to 6.5±1.4 mol/min per kg fat-free mass [FFM] (P<0.05) and during HT it was completely suppressed. In contrast, R _ox increased to 152±21 and 61±10 mol/min per kg FFM at the end of HT and ET respectively (P<0.05). HT (i. e., plasma glucose 10 mM) and hyperinsulinaemia (24.5±0.9 U/ml) also increased total carbohydrate oxidation from 203±7 (ET) to 310±3 mol/min per kg FFM (P<0.0001) and suppressed fat oxidation from 51±3 (ET) to 18±2 mol/min per kg FFM (P<0.0001). As the rates of oxidation at more physiological euglycaemic concentrations of glucose were limited to 92±9 mol/ min per kg FFM, and were similar to those reported when carbohydrate is ingested, the results of the current study suggest that the concentrations of glucose and insulin normally present during prolonged, intense exercise may limit the rate of muscle glucose uptake and oxidation.     

Acetylcholine-stimulated changes of membrane potential and intracellular Ca2+ concentration recorded in endothelial cells in situ in the isolated rat aorta

The intracellular free Ca²⁺ concentration and membrane potential changes evoked by acetylcholine were recorded from whole-cell patch-clamped endothelial cells in situ in the isolated rat aorta. The endothelium had a resting membrane potential of –52±3 mV (SEM, range –35 mV to –76 mV n=34) and a low input resistance (32–54 M). The membrane potential hyperpolarised by 3–30 mV on continuous application of acetylcholine at concentrations that produced endothelium-dependent relaxations in isolated rat aortic rings (range 1–500 nM). The response often comprised complex fluctuations of hyperpolarised membrane potential. Calcium concentration was measured with the fluorescent indicator furaptra, which has a wide range and minimises Ca²⁺ buffering. Acetylcholine evoked an initial rapid elevation of intracellular Ca²⁺ concentration, peaking in the range 6–35 M, which declined with a half time of approximately 6 s, followed by repetitive [Ca²⁺] spikes of amplitude 2–18 M in 23 of 34 cells. The initial [Ca²⁺] transient and hyperpolarisation were unaffected by removal of external Ca²⁺, whilst subsequent [Ca²⁺] spikes and maintained hyperpolarisations required the presence of external Ca²⁺. Both the hyperpolarisation and Ca²⁺ responses elicited by acetylcholine were abolished by atropine (1 M). These results show that endothelial cells in situ exhibit large, fast repetitive [Ca²⁺] spikes in response to extracellular acetylcholine.     

The effects of pressure on the water permeability of the descending limb of Henle's loops of rabbits

Descending limbs of Henle's loops from rabbits were perfused in vitro. Using techniques where the collecting pipets permitted cannulation of the tubule, we were able to maintain reasonable flow rates at lower perfusion reservoir heights than are required with a conventional Sylgard seal pipet. The bath was either isosmotic to the perfusate, or was made 300 mOsm hyperosmotic using urea. Net water reabsorption did not occur in tubules perfused at low pressure (average reservoir height = 26 cm H₂O) even when the bath was hyperosmotic: J _v =–0.06 ±0.18 nl/min (n=7). Observed increases in sodium concentration and osmolality of collected fluid, when the bath was made hyperosmotic, were 16±8 mM (n=7) and 254±38 mOsm (n=7), respectively. Presumably the large increase in osmolality of the collected fluid was due to entrance of urea.When the Sylgard seal collecting end was utilized higher perfusion reservoir heights had to be used to maintain flow (mean height 66 cm H₂O). These tubules were highly permeable to water as reported by others for this tubule segment. In the presence of a hyperosmotic bath water extrusion resulted in a dramatic increase in the osmolality of the collected fluid (312 ±5 mOsm; 7 tubules) which was almost completely accounted for by an increase in sodium concentration (153±8 mmole/l; 6 tubules).The¹⁴C urea permeability (measured lumen to bath) of descending limbs in a 300 mOsm bath was 0.64 ×10^–7 cm²·s^–1±0.23×10^–7 (11 tubules). When the bath was made hyperosmotic using urea or raffinose the¹⁴C urea permeability increased significantly.     

Oxidation of 14C-β-hydroxybutyrate administered by ventriculo-cisternal perfusion in the dog

Summary A ventriculo-cisternal perfusion system (from the 3rd ventricle to cisterna magna) was employed to study the oxidation of Na-DL-3-hydroxybuty-rate-3-¹⁴C when added to the perfusion fluid. ¹⁴CO₂ appeared in the perfusate and in the blood taken from both the confluens sinuum and a peripheral artery, in the course of the perfusion. Specific activity of CO₂ in the perfusate usually exceeded that of the venous blood, which in turn was always higher than that of arterial blood. The calculated rate of oxidation of OHB to CO₂ was about 20 mmole/min, or 5–6% of the perfused OHB. It is postulated that OHB supplied locally in CSF may serve some supplemental physiological role as an oxidizable substrate.Supported by Grant HE 03130 from the National Heart Institute.     

Inhibition of chemiluminescence in granulocytes and alveolar macrophages by azelastine

The effect of azelastine, an orally effective antiasthmatic/antiallergic drug, on the generation of oxygenderived free radicals in phagocytes was investigated using different chemiluminescence-assays. The chemiluminescence (CL) of both human polymorphonuclear granulocytes (PMNL) and guinea-pig alveolar macrophages (AM) was induced either by phorbol myristate acetate (PMA) or zymosan and amplified either by lucigenin or DMNH (7-dimethylamino-naphthalene-1,2-dicarbonic-acidhydrazide). The inhibitory effect of azelastine was dependent on the inducer employed and the condition and type of cells used. Azelastine reduced PMA-induced CL concentration-dependently in both PMNL (IC₃₀=3.9 M) and AM (IC₃₀=9.8 M). In AM zymosan-induced CL was inhibited 21.7% by 10 M azelastine, whereas in PMNL it remained unchanged up to 10 M azelastine. Azelastine has a significantly stronger inhibitory effect (IC₃₀=4.2 M) on oxygen free radical generation in AM primed by fetal calf serum than in unprimed AM. Based on present results it is likely that azelastine inhibitis oxygen-derived free radical generation by interaction with protein kinase C.     

Effects of ryanodine on skinned skeletal muscle fibers of the rabbit

The mechanism(s) of ryanodine-induced contracture of skeletal muscle were studied in skinned fibers from soleus (SL) and adductor magnus (AM) (slow- and fast-twitch skeletal muscles) of rabbits. Pieces of SL or AM were homogenized (sarcolemma disrupted). Single fibers were dissected from the homogenate and mounted on photodiode force transducers. At concentrations 1–50 M, ryanodine slightly but significantly increased the submaximal Ca²⁺-activated tension development of the contractile proteins in skinned fibers of AM but not of SL. Ryanodine in uptake phase or release phase increased caffeine-induced tension transients in the SR of both muscle types; however, no dose-response relation was found. Ryanodine 1 M decreased, however, the second control tension transients in a dose-dependent manner. The depression was nearly irreversible and activity-dependent. The concentrations of ryanodine that inhibited the second control tension transients by 50% were 10 M and 5 M for SL and AM, respectively, following ryanodine administration in the release phase, and 100 M and 30 M, respectively, for these preparations after the drug was present in the uptake phase. The quantity of calcium released from the SR by Triton X-100 and caffeine in the second control tension transient was unchanged by ryanodine at all concentrations tested when compared with that of the absence of ryanodine. The present findings suggest that the ability of ryanodine to increase immediate calcium release from the SR, and in AM but not SL, to increase the sensitivity of the contractile proteins to Ca²⁺ underlies the contracture caused by this agent in intact skeletal muscles. The delayed decreased Ca²⁺ efflux by caffeine, as evidenced by depression of tension transient with no change in the calcium content may be responsible for the decreased twitch tension caused by this agent.     

Exclusive asymptomatic neonatal infections by human rotavirus strains having subgroup I specificity and “long” RNA electropherotype

Summary A large number of stool specimes, of healthy new born infants, collected from various hospitals and clinics in Bangalore City, India, have been examined for the presence of asymptomatic rotaviral excretion. Out of 370 samples analysed during a three year period from 1988 to 1991, 133 specimens (36%) were positive for rotavirus RNA. All these asymptomatic neonatal strains, without exception, showed long RNA pattern, but subgroup I specificity. Serotype analysis by ELISA or by hybridization with serotype-specific probes indicated that these strains probably represent a new serotype in newborn children. We find an exclusive association of human rotaviruses having long RNA pattern and subgroup I specificity with asymptomatic neonatal infections in contrast to the earlier observations of association of such unusual strains with acute gastroenteritis in young children.     

Tension development and calcium sensitivity in skinned muscle fibres of the frog

Calcium activated isometric tension development was measured in single skinned muscle fibres of the ileofibularis muscle of the frog. The experiments were carried out at 5°C, pH=6.9, 1 mM free Mg²⁺ and an ionic strength of 160 mM. A Hill curve was fitted to the isometrically developed tension at different Ca²⁺ concentrations by means of a non-linear least mean square approximation. At a sarcomere length of 2.15 m, the Ca²⁺ concentration for half maximum tension (K) was 1.6 M. This Ca²⁺ concentration decreased with increasing sarcomere length; at 2.7 m, K was 1.1 M and at 3.1 m, K was 0.9 M. Therefore, Ca sensitivity is increased at larger sarcomere lengths. Consequently, the optimal sarcomere length for tension development shifted to larger values when the Ca²⁺ concentration was lowered. Osmotic compression of the fibre at 2.15 m by means of 5% Dextran also caused an increase in Ca sensitivity (K was 1.0 M). At 2.7 m, addition of 5% Dextran hardly affected the Ca sensitivity. The possible role of the interfilament spacing in the explanation of these results discussed.     

Inhibition of L-type calcium channels by internal GTP [γS] in mouse pancreatic β cells

Pretreatment of pancreatic cells with pertussis toxin resulted in a 30% increase in peak whole-cell Ca²⁺ currents recorded in the absence of exogenous intracellular guanine nucleotides. Intracellular application of 90 M GTP[S], by liberation from a caged precursor, resulted in 40% reduction of the peak Ca²⁺ current irrespective of whether the current was carried by Ca²⁺ or Ba²⁺. Effects on the delayed outward K⁺ current were small and restricted to a transient Ca²⁺-dependent K⁺ current component. Inhibition by GTP[S] of the Ca²⁺ current was not mimicked by standard GTP and could not be prevented either by pretreatment with pertussis toxin or by inclusion of GDP[S] or cyclic AMP in the intracellular medium. The inhibitory effect of GTP[S] could be counteracted by a prepulse to a large depolarizing voltage. A similar effect of a depolarizing prepulse was observed in control cells with no exogenous guanine nucleotides. These observations indicate that inhibition of cell Ca²⁺ current by G protein activation results from direct interaction with the channel and does not involve second-messenger systems. Our findings also suggest that the cell Ca²⁺ current is subject to resting inhibition by G proteins.     

Modulation of Ca2+-activated K+ channels by Mg2+ and ATP in frog oxyntic cells

Ca²⁺-activated K⁺ channels in the basolateral plasma membrane of bullfrog oxynticopeptic cells are intimately involved in the regulation of acid secretion. Patch-clamp techniques were applied to study the regulating mechanism of these channels. In the excised inside-out configuration, intracellular Mg²⁺ decreased channel activity in a dose-dependent manner. In the absence of Mg²⁺, administration of adenosine 5 triphosphate (ATP) to the cytoplasmic side also inhibited channel activity. On the other hand, in the presence of Mg²⁺, addition of ATP markedly increased channel activity. At a fixed concentration of free Mg²⁺ the Mg-ATP complex caused channel activation and shifted the dose response relationship between channel activity and the intracellular Ca²⁺ concentration to the left. A nonhydrolysable ATP analogue, adenosine 5-[,-imido]triphosphate (AMP-PNP) adenylyl [,-methylene]diphosphate (AMP-PCP), could not substitute for ATP in channel activation, but a hydrolysable ATP analogue, adenosine 5-O-(3-thiotriphosphate) (ATP[S]) could do so. Furthermore, application of alkaline phosphatase to the cytoplasmic side inhibited channel activity. These results demonstrate that Ca²⁺-activated K⁺ channels are regulated by Mg²⁺ and ATP, and suggest that a phosphorylation reaction may be involved in the regulation mechanism of these channels.     

The distribution of membrane bound enzymes in the acini and ducts of the cat pancreas

Summary Enzymes activities of the Na⁺K⁺-and the HCO₃ ^–-ATPases, alkaline phosphatase, amino peptidase and 5 nucleotidase have been measured in 4 different preparations from the cat pancreas a) in the ducts including all sizes b) in ducts of three different diameters c) in that tissue, which had been dissected off from the ducts, called acini, and d) in the whole homogenate of the pancreas. The distribution of the measured enzymes shows, that the Na⁺K⁺-activity is highest in the acinar structures (mean value 0.532 M/mg Protein x h), while the ducts show nearly no Na⁺K⁺-ATPase activity. The HCO₃ ^–-ATPase, the alkaline phosphatase and the 5 nucleotidase are in the ducts between 2.4 and 3.6 times higher than in the whole organ whereas the amino peptidase does not appear to have a selective distribution. As the HCO₃ ^–-ATPase activity distribution pattern is identical with that of the secretory capacity of HCO₃ ^– as evidenced by earlier micropuncture studies, the data suggest that the HCO₃ ^–-ATPase is the main enzyme involved in the secretion of the bicarbonate buffer. Concerning the Na⁺K⁺-ATPase activity in the acinar structures we cannot contribute to its function in the enzyme secreting process.