Functional compartmentalization of energy production in neural tissue

Previous work in our laboratory has shown that neural trauma results in a disparity between oxidative and glycolytic rates. In non-neural tissue, glycolysis and oxidative phosphorylation have been shown to work independently of one another, a phenomenon known as "energy compartmentalization". We believe that functional compartmentalization of energy production may also occur in the brain with glycolysis providing energy for membrane bound ionic pumps. Spreading depression, induced in rodent brain by topical KCl application, results in K+ shifts. The restoration of K+ gradients is accomplished by energy dependent Na(+)-K+ pumps. If these pumps depend upon glycolysis, blocking glycolysis should prevent reconstitution of normal [K+]e levels. The present series of experiments were designed to suggest that energy compartmentalization may also exist in brain, and that glycolytic energy production is preferentially used by Na(+)-K+ pumps to maintain normal ionic homeostasis by observing the dynamics of spreading depression induced K+ shifts before and after glycolytic blockade. Spreading depression was associated with increased K+ (48.6 +/- 16.6 mM over control) that normalized within 2.9 +/- 0.3 minutes. Following superfusion with a glycolytic blocking agent, spreading depression produced similar increases in [K+]e (40.6 +/- 12.0 mM over control) but time for reconstitution of the normal [K+]e was 400% longer than controls (2.9 +/- 0.3 to 14.9 +/- 2.1 minutes, P less than 0.001). Time required for recovery of EEG was identical pre- and post-blockade. We believe these data suggest that energy compartmentalization may exist in neural tissue and that glycolytic pathways of energy production are functionally tied to membrane Na(+)-K+ pumps.(ABSTRACT TRUNCATED AT 250 WORDS)     

The Drosophila Mutant tetanic Interacts with a Gene Complex Including the Structural Locus of K+ Channels and Shows Altered Dephosphorylation and Learning

The tetanic (tta; X.-52.6) mutation has been isolated on the basis of its sensitivity to extradoses of the normal Shaker gene complex (ShC) where the K+ channel la is encoded. The mutant shows up to threefold elevation of the membrane bound protein phosphatase type 1 (PP1) activity in body extracts, probably due to reduced levels of the PP1 specific inhibitor 2 (I-2). By contrast, PP1 activity in the head is only half of the normal value. In addition, tta fails to perform normally in a negative reinforcement olfactory paradigm. The functional relationships between phosphorylation, K+ currents, phosphatase activity and modulation of synaptic activity during learning and memory are discussed in the light of their possible genetic links.     

Synthetic peptides corresponding to the sequence of noxiustoxin indicate that the active site of this K+ channel blocker is located on its amino-terminal portion

Summary A nonapeptide Thr-Ile-Ile-Asn-Val-Lys-Cys-Thr-Ser (NTX_1–9) and a decapeptide Met-Asn-Gly-Lys-Cys-Lys-Cys-Tyr-Asn-Asn (NTX_30–39) corresponding to the N-terminal and C-terminal sequences respectively of Noxiustoxin (NTX) were synthesized by the solid phase method of Merrifield (1963). The first synthetic peptide (NTX_1–9) was shown to be toxic to mice independently of the route of administration: intraperitoneally, subcutaneously or intraventricularly (100–200 g/20 g mouse weight). The second (NTX_30–39) was not toxic even at higher dose (400 g/20 g mouse). When the effects of the peptide NTX_1–9 and of the authentic toxin (Noxiustoxin) were studied on the liberation of [³H] 4-aminobutyric acid (³H-GABA) from mouse synaptosomes, both gave essentially the same results, except that peptide NTX_1–9 was needed at higher concentration. Synthetic peptide NTX_30–39 had no effect in the same preparation at even higher doses. The GABA release produced by toxic peptide NTX_1–9 was not affected by tetrodotoxin but was completely abolished by the presence of the K⁺ ionophore valinomycin, mimicking the effect of native NTX in the same system (Sitges et al., 1986). These results indicate that the toxic active site of Noxiustoxin is possibly located in or near the N-terminal amino acid portion of the molecule.Abbreviations used BOC amino acids ter-butyloxycarbonyl-amino acids - BOC amino acid-PAM-resin ter-butyloxycarbonyl-aminoacyl-4-(oxymethyl)-phenacetamidomethyl-resin - GABA 4 aminobutyric acid - HPLC high performance liquid chromatography - MSA mouse serum albumin - NTX Noxiustoxin - NTX _(numbers) synthetic peptides with amino acid sequences of NTX at position start (first number) to position end (second number) of the sequence according to Fig. 1 - TTX tetrodotoxin Supported in part by the Mexican Council of Science and Technology (CONACyT), grants PVT/QF/NAL/84/2182, PVT/AI/NAL/85/3029 to L.D.P.; PCSACNA 022640 to A.B. A patent request claiming rights on the use of synthetic NTX and related peptides was presented in Washington, DC (U.S.A.), Serial number 07/132,169, filing date December 14, 1987.     

Cardiovascular effects of endothelin 1 in pithed spontaneously hypertensive rats: evaluation of its mechanism(s) of action

Summary The present experiments were carried out to investigate the cardiovascular effects of endothelin 1 (ET) in pithed spontaneously hypertensive (SH) rats and to evaluate its mechanism of action. The results show that ET (0.1 – 3 nmol/kg i.v.) is a powerful vasoconstrictor agent in the pithed rat. However, at a dose of 3 nmol/kg i.v. all the pithed animals died following a gradual decrease in mean arterial blood pressure and pulse pressure and changes in the form of the electrocardiogram (ECG). The predominant feature of the change in the ECG was a progressive decrease in the amplitude of the T wave resulting in a depression of the curve representing repolarization. Investigations in isolated perfused SH rat hearts showed that ET powerfully reduces coronary flow concentration-dependently (IC₅₀ 2.1 ±0.3 nM) an effect associated with sinus bradycardia and a decrease in coronary pressure amplitude. No overt ECG changes were seen. Control experiments with mechanical flow restriction suggest that bradycardia is a consequence of reduced coronary flow and that the ECG changes observed in vivo can be explained on the basis of coronary insufficiency and resulting myocardial hypoxia. Vasoconstrictor responses to angiotensin II (0.4 g/kg i.v.), phenylephrine (8 g/kg i.v.) and ET (0.5 nmol/kg i.v.) were antagonised by around 70% by isradipine (0.03 mg/kg i.a.). The results suggest that endothelin-induced vasoconstriction may involve receptor operated channel activation and opening of voltage sensitive Ca²⁺ channels.Send offprint requests to A. K. Mir at the above address     

Inhibitory effect of amiloride and gadolinium on fine afferent nerves in the rat knee: evidence of mechanogated ion channels in joints

Synovial joints are complex sensory organs which provide continuous feedback regarding position sense and degree of limb movement. The transduction mechanisms which convert mechanical forces acting on the joint into an electrochemical signal which can then be transmitted to the central nervous system are not well understood. The present investigation examined the effect of the mechanogated ion channel blockers amiloride and gadolinium on knee joint mechanosensitivity. In deeply anaesthetised rats (sodium thiopental: 100–120 mg/kg, i.p.), single unit extracellular recordings were made from knee joint group III (Aδ) and group IV (C) primary afferents in response to mechanical rotation of the joint. Afferent firing rate was measured before and after topical application of either amiloride (0.1 mM, 1 mM) or gadolinium (250 μM) onto the receptive field of the sensory unit and recording was continued every 10 min up to a total of 50 min. With normal rotation of the knee, joint mechanosensitivity was significantly reduced by both amiloride (P<0.0001; n=10–21) and gadolinium (P=0.001; n=12) and this effect was sustained throughout the recording period. This investigation provides the first in vivo electrophysiological evidence that joint mechanotransduction involves the activation of amiloride and gadolinium-sensitive mechanogated ion channels. Future studies to determine the mechanogated ion channel subtypes present in joints and the modulation of their gating properties during inflammation may yield novel approaches for the control of arthritis pain. Funding: JJMcD is funded by the Alberta Heritage Foundation for Medical Research, the Canadian Institutes for Health Research, and the Arthritis Society of Canada.     

Effect of papaverine on tone and contractions of the depolarized smooth muscle of the guinea pig taenia coli

Experiments were carried out on isolated strips of guinea pig taenia coli. The smooth muscle was depolarized in a solution with high potassium concentration (120 mM KCl). The effect of papaverine (in concentrations of 10^–5 to 3.10^–5 g/ml) on the tone and off-response to a prolonged and strong hyperpolarizing current was investigated on the denervated muscle. Papaverine was found: 1) to abolish contractile responses to application of histamine, bradykinin, and acetylcholine; 2) to reduce the tone of the depolarized muscle and abolish the effect of an increase in the Ca⁺⁺ concentration in the external medium on muscle tone; 3) to have no effect on the amplitude and velocity of the ascending phase of the off-response; 4) to accelerate the descending phase of the off-response. The following hypotheses are put forward to explain the result: 1) in the cell membrane there are chemically excitable calcium channels which are blocked by papaverine; 2) in the membrane there are calcium leakage channels responsible for the maintenance of tone and blocked by papaverine; 3) papaverine has negligible effect on electrically excitable calcium channels.A. V. Vishnevskii Institute of Surgery, Academy of Medical Sciences of the USSR, Moscow. Bio-Inorganic Chemicals BIKhS Research Institute, Kupavna, Moscow Region. (Presented by Academician of the Academy of Medical Sciences of the USSR N. A. Fedorov.) Translated from Byulleten' Éksperimental'noi Biologii i Meditsiny, Vol. 85, No. 2, pp. 177–180, February, 1978.     

Shear stress induced membrane currents and calcium transients in human vascular endothelial cells

We have measured membrane currents induced by shear stress together with intracellular calcium signals in endothelial cells from human umbilical cord veins. In the presence of extracellular calcium (Ca²⁺]_o), shear stress induced an inward current at a holding potential of 0 mV which is accompanied by a rise in intracellular Ca²⁺ ([Ca²⁺]_i). In the absence of extracellular calcium shear stress was unable to evoke a calcium signal but still induced a membrane current. The voltage dependence of the shear stress induced current was obtained from difference currents evoked by linear voltage ramps before and during application of shear stress. Its reversal potential E_rev shifted from –2.3±0.8 mV (n=4) in a nominally Ca²⁺ free solution to +1.5±1.6 mV at 1.5 mM [Ca²⁺]_o (n=4) and to +21.9±4.4 mV (n=7) at 10 mM [Ca²⁺]_o. From our data we conclude that shear stress opens an ion channel that is 12.5±2.9 (n=7) times more permeable for calcium than for sodium or cesium.     

Ensemble noise and current relaxation analysis of K+ current in single isolated salivary acinar cells from rat

The K⁺ channel in rat parotid gland acinar cells were investigated by ensemble current noise analysis in single isolated cells employing the giga-seal whole cell current recording mode. Sets of 20–40 identical de- and hyperpolarization voltage steps were applied and the resultant current records were processed by computer to obtain the mean and the variance of the current. The time-course of the mean current could be fitted by the sum of two exponentials, suggesting a 3-state model. The simplest plausible hypothesis is a model with one open and two closed states. Assuming this model, the relationship between the variance (₂) and the mean current (I) could be fitted by the function ₂/I=i–I/N. The estimated single channeli/V-relations were similar to those taken from single channel current recordings, and the size of the population of channels per cell (N) was 76±26 (n=12). The validity of the model was tested by a successful simulation of the time-course of the variance.     

Channels in planar bilayers made from commercially available lipids

Planar bilayer membranes were made from commercially available lipids. With various lipids, originating from biological sources, such as egg yolk phosphatidylcholine, soybean phosphatidylcholine or bovine brain phosphatidylserine, spontaneously fluctuating channels could be observed. Spontaneous channel activity could not be observed in planar bilayers made from synthetic lipids, such as 1,2 diphytanoyl 3-sn-phosphatidylcholine. It is concluded that the observed channels are due to proteolipids, being present as impurities in the lipid material.