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PubMed1970

Membrane properties of a barnacle photoreceptor examined by the voltage clamp technique.

1. Electrical properties of the membrane of photoreceptor cells in the lateral ocelli of barnacles, Balanus amphitrite and B. eburneus were investigated by intracellular recording, polarization and voltage-clamp techniques.2. The resting potential of a dark adapted cell was 36.3 +/- 6.6 mV (S.D.) and depended mainly on the external K(+) concentration.3. Current-voltage relations obtained from voltage-clamp experiments in the absence of light were non-linear and varied with time after the onset of a step change in membrane potential; the steady state was reached after about 0.5 sec.4. Illumination resulted in a membrane potential change under current clamp and in a change of membrane current (light-initiated membrane current (L.I.C.): total membrane current with illumination minus current without illumination) under voltage-clamp conditions. Amplitudes and time course of L.I.C. depended on the light intensity as well as membrane potential.5. The L.I.C.-voltage relation was non-linear and corresponded with a slope conductance increase with increasing positive membrane potential.6. The reversal potential of L.I.C. was independent of the light intensity and the time after onset of illumination; the average value obtained in normal saline was +26.9 +/- 5.0 mV.7. The membrane conductance estimated from instantaneous L.I.C.-voltage relations agreed with the chord conductance of the non-linear L.I.C.-voltage relation.8. Decreasing external Na(+) concentration decreased the inward component of L.I.C. but not the outward component.9. Decreasing external Ca(2+) concentration increased the inward as well as the outward component of L.I.C.10. The reversal potential shifted in the negative direction with decreasing external Na(+) concentration (the rate was 10-15 mV for a tenfold change in concentration) and the rate was augmented in the absence of Ca(2+) but did not exceed 21 mV.11. The change of reversal potential with changes of external Ca(2+) concentration was negligible in normal Na(+) media but was significant in the absence of Na(+) (rate as high as 20 mV).12. Alteration of the external K(+) or Cl(-) concentrations did not affect the amplitude or reversal potential of L.I.C.13. The results indicate that illumination increases the membrane permeability mainly to Na(+) ions and that the primary effect of Ca(2+) ions is suppression of the permeability increase; Ca(2+) permeability may increase slightly during illumination.

Animals↗
PubMed1975

Effects of stimulating the acetylcholine receptor on the current-voltage relationships of the smooth muscle membrane studied by voltage clamp of potential recorded by micro-electrode.

1. A double sucrose-gap voltage-clamp technique is described for use on smooth muscle strips longer than about 2 mm. It involves intracellular recording by microelectrode of the membrane potential of a narrow region of the strip ("node") sandwiched between two streams of deionized sucrose solution. Current was passed into the node across one or both sucrose streams. 2. Preliminary experiments in which potential was recorded intracellularly at two points during polarization of a "short cable" preparation, formed by folding over a strip of smooth muscle, suggested that a node width of less than 0-15 mm was needed to achieve uniform potential during inward current flow. However, when node width between sucrose-gaps was reduced to 0-5 mm, spontaneous electrical activity was lost, and below 0-5 mm spike threshold was raised and the regenerative spike became graded. The currents flowing during the application of rectangular voltage-clamp command potentials were described. 3. Using taenia smooth muscle it was shown by recording with a second, independent micro-electrode that potential was not uniform for up to 200 ms or more following a step change in potential under voltage-clamp in nodes 0-4-0-5 mm wide where current was passed across both sucrose gaps. However, reasonably uniform nodal potentials were obtained using ramps with relatively slow rates of rise (25 mV/s). 4. Using such slow ramp commands under voltage clamp, the effects of carbachol on the current-voltage relationship of longitudinal muscle of ileum and taenia were studied in hypertonic solution. 5. In the presence of carbachol (10(-6) to 10(-5) g/ml.) additional inward current flowed across the membrane (in some experiments an equilibrium potential was observed at which this current reversed direction). The magnitude of this additional current was linearly related to potential at potentials negative to the resting potential. At potentials positive to the resting membrane potential, this additional current increased with depolarization over the range -40 to -10 mV; in ileum the effect of this additional inward current on the current-voltage relationship was to produce a region of net inward current where before, in the absence of carbachol, a net outward current existed. In taenia the additional inward current flowing in the presence of carbachol was too small to produce a region of net inward current; thus carbachol produced regenerative slow oscillations of potential (slow waves) in ileum but not in taenia. 6. These results support a previous suggestion that activation of the acetylcholine receptor of ileal smooth muscle produces an additional inward current in the membrane which increases with depolarization and is responsible for the regenerative slow waves seen when muscarinic stimulants are applied. A similar effect apparently operates in taenia but the additional inward current is too small to produce regenerative slow waves.

Animals↗
PubMed1977

Sodium efflux from voltage clamped squid giant axons.

1. The efflux of radioactive sodium was measured from squid axons during simultaneous voltage clamp experiments such that it was possible to determine the efflux of sodium associated with a measured voltage clamp current. 2. The extra efflux of sodium associated with voltage clamp pulses increased linearly with the magnitude of the depolarization above 40 mV. A 100 mV pulse of sufficient duration to produce all of the sodium current increased the rate constant of efflux by about 10(-6). 3. Application of 100 nM tetrodotoxin eliminated the sodium current and the extra efflux of radioactive sodium. 4. Cooling the axon increased the extra efflux/voltage clamp pulse slightly with a Q10 of 1/1-1. On the same axons cooling increased the integral of the sodium current with a Q10 of 1/1-4. 5. Replacing external sodium with Tris, dextrose or Mg-mannitol reduced the extra efflux of sodium by about 50%. The inward sodium current was replaced with an outward current as expected. 6. Replacing external sodium with lithium also reduced the extra efflux by about 50% but the currents seen in lithium were slightly larger than those in sodium. 7. The effect of replacing external sodium was not voltage dependent. Cooling reduced the effect so that there was less reduction of efflux on switching to Tris ASW in the cold than in the warm. 8. The extra efflux of sodium into sodium-free ASW is approximately the same as the integral of the sodium current. Adding external sodium produces a deviation from the independence principle such that there is more exchange of sodium than predicted. Such a deviation from prediction was noted by Hodgkin & Huxley (1952c). 9. Using the equations of Hodgkin & Huxley (1952c) modified to include the deviation from independence reported in this paper and its temperature dependence, one can predict the temperature dependence of the sodium efflux associated with action potentials and obtain much better agreement than is possibly without these phenomena. 10. This deviation from independence in the sodium fluxes is the type expected from some kind of mixing and binding of sodium within the membrane phase.

Animals↗
PubMed1982

Voltage-clamp studies of transient inward current and mechanical oscillations induced by ouabain in ferret papillary muscle.

1. We studied the effects of a toxic concentration of ouabain on transmembrane electrical activity and on mechanical behaviour of right ventricular papillary muscles from ferrets in a single sucrose-gap using current clamp and voltage clamp.2. Ouabain (1.4-1.8 muM) induced oscillatory after-potentials and after-concentrations in current-clamp experiments. Voltage clamp showed that the oscillatory after-potential was caused by a transient inward current, similar to that in Purkinje fibres.3. The transient current had a sigmoidal dependence on the preceding (activating) voltage step V1, with a treshold around -13 mV and a plateau between +10 and 20 mV. There was a decline in current amplitude for more positive clamps. When activated by a fixed V1 voltage step, and measured at different repolarization levels V2, the transient current manifested an inverse dependence on V2 between -50 and -10 mV. No outward transient current could be detected. Total replacement of Na in the bathing medium by Tris or by sucrose abolished the transient current.4. Ouabain caused an increase of phasic (twitch) tension responses to voltage steps at all potentials without shifting the curve relating these variables on the voltage axis. The drug evoked an even greater increase in the tonic tension responses.5. After prolonged exposure, oscillatory mechanical responses were frequently recorded during positive voltage steps. Unlike the after-contraction, these mechanical fluctuations were not consistently damped and were not accompanied by detectable synchronous current fluctuations. Catecholamines and dibutyryl cyclic AMP markedly reduced the amplitude of the tonic contraction and the mechanical oscillations but increased their frequency. Caffeine had no effect on the tonic contraction amplitude but abolished the fluctuations.6. These results support the proposal that Ca is transiently released from the overloaded sarcoplasmic reticulum in ouabain-intoxicated muscle and may evoke oscillatory responses in nearby contractile fibrils. When these transient increases of sarcoplasmic free Ca are large enough, they may induce the transient transmembrane current described above.

Animals↗
PubMed1982

The passive electrical properties of guinea-pig ventricular muscle as examined with a voltage-clamp technique.

1. A voltage-clamp technique was developed for stable recording of small currents in guinea-pig ventricular muscle. Small cylindrical preparations were impaled with three micro-electrodes, one for measuring the feed-back potential and two for injecting current. 2. The longitudinal potential profile resulting from current injection at one point was measured. It agreed well with the theoretical predictions for a linear cable which is sealed at both ends ('healing over'), with a length constant (lambda) of 580 +/- 145 micron. 3. When the clamp current was injected symmetrically into each half of the preparation via two electronic current pumps a spatially homogeneous clamp could be achieved in preparations with a diameter of less than or equal to 250 micron and a length of less than or equal to 2 lambda. 4. The membrane capacity and the membrane resistance of the preparations at the resting potential were measured with small voltage-clamp pulses. Assuming a specific membrane capacity (Cm) of 1 microF/cm2 a specific membrane resistance (Rm) of 6.7 +/- 1.8 k omega cm2 was obtained in Tyrode solution containing 3 mM-K. 5. The total surface area was calculated from the measured capacity of the preparation assuming a Cm of 1 microF/cm2. The total cellular volume was estimated from optical measurement of the external dimensions of the preparation assuming an extracellular space of 25%. From these data the average surface/volume ratio of individual cells was calculated to be 7200 cm2/cm3. 6. From the measured electrical constants the specific resistance of the intracellular space (Ri) was calculated to be 200-250 omega cm. With small constant current pulses a membrane time constant of 6.6 +/- 1.3 ms was measured. 7. The influence of the extracellular potassium concentration ([K]o) on Rm was studied in the range 1.5-6 mM-[K]o. Rm was found to depend on [K]o less than predicted by the constant field theory.

Animals↗
PubMed1987

Alpha-adrenergic inhibition of rat cerebellar Purkinje cells in vitro: a voltage-clamp study.

1. The effects of the alpha 2-adrenergic agonist clonidine on the membrane properties of Purkinje cells were analysed in sagittal slices of adult rat cerebellum by the use of intracellular recordings performed at a somatic level in the single-electrode voltage-clamp mode. 2. In preliminary current-clamp experiments, clonidine elicited in all cells a hyperpolarization 3-8 mV in amplitude, accompanied by a 15-35% increase of the input resistance when it was added to the bath at a concentration of 2-5 microM. 3. In voltage-clamped cells at a potential of -65 mV. the same concentration of clonidine always induced an outward shift of the holding current (0.2-0.5 nA in amplitude), thus corresponding to the hyperpolarization seen in current-clamp experiments, and this effect was accompanied by a clear increase of membrane resistance. Furthermore, clonidine markedly depressed the inward relaxations induced by hyperpolarizing commands of amplitude less than 10-20 mV whereas those induced by larger steps were much less affected. All these effects of clonidine were reversible when the drug was washed out. 4. When the slices were bathed in a medium containing 10 mM-Cs and 5 X 10(-6) M-tetrodotoxin, the inward relaxations induced by hyperpolarizing steps were abolished. However, a small inward current was still present when the membrane potential was stepped back to -65 mV, which was in turn blocked by the Ca-channel blocker Cd. This inward Ca current was also blocked by 2-5 microM-clonidine in the bath. 5. All these effects of clonidine were abolished by the alpha 1-adrenergic antagonists prazosin and phentolamine at concentrations of 0.5 and 40 microM respectively in the bath. In contrast, they were only weakly antagonized or unaffected by 2 microM of the alpha 2-adrenergic antagonist yohimbine. 6. On the basis of these results and of a previous work on the ionic basis of the inward rectification of Purkinje cells (Crepel & Penit-Soria, 1986), it appears that these neurones exhibit a well developed alpha (possibly alpha 1)-adrenergic inhibition of a low-threshold Ca conductance and a Ca-dependent K conductance operating near resting potential.

Adrenergic alpha-Agonists↗
PubMed1988

Plateau and descending limb of the sarcomere length-tension relation in short length-clamped segments of frog muscle fibres.

1. The relation between sarcomere length and tetanic tension was determined at 10-12 degrees C for 70-80 microns long segments of single fibres isolated from the tibialis anterior and semitendinosus muscles of the frog. Measurements of segment striation spacings were performed during fixed-end or length-clamp contractions by means of a laser light diffractometer. 2. At sarcomere lengths of around 2.10 microns tetanic tension rose promptly to a steady plateau, independent of the recording conditions. At greater sarcomere lengths under fixed-end conditions the tension rise occurred in two distinct stages: an initial rapid rise followed by a much slower creep. The tension creep was entirely abolished in length-clamp contractions. 3. The sarcomere length-tension diagram of length-clamped segments of tibialis anterior fibres exhibited a definite flat region between about 1.96 and 2.16 microns where tension varied by less than 1.5%. The highly linear descending limb reached zero tension at about 3.53 microns. The shift to the left by about 0.10 microns, with respect to the length-tension diagram of length-clamped segments of semitendinosus fibres, may be tentatively explained by assuming that thin filament lengths vary in different muscles. 4. The results are in agreement with those of a previous work by Gordon, Huxley & Julian (1966) and support the hypothesis (Huxley, 1957, 1980) that muscle tension is produced by simultaneous action of independent force generators, in proportion to the number of myosin bridges overlapped by actin filaments.

Animals↗
PubMed2000

Probing the interaction of the cytotoxic bisdioxopiperazine ICRF-193 with the closed enzyme clamp of human topoisomerase IIalpha.

Topoisomerase II is an ATP-operated protein clamp that captures a DNA helix and transports it through another DNA duplex, allowing chromosome segregation at mitosis. A number of cytotoxic bisdioxopiperazines such as ICRF-193 target topoisomerase II by binding and trapping the closed enzyme clamp. To investigate this unusual mode of action, we have used yeast to select plasmid-borne human topoisomerase IIalpha alleles resistant to ICRF-193. Mutations in topoisomerase IIalpha of Leu-169 to Phe (L169F) (in the N-terminal ATPase domain) and Ala-648 to Pro (A648P) (in the core domain) were identified as conferring >50-fold and 5-fold resistance to ICRF-193 in vivo, respectively. The L169F mutation, located next to the Walker A box ATP-binding sequence, resulted in a mutant enzyme displaying ICRF-193-resistant topoisomerase and ATPase activities and whose closed clamp was refractory to ICRF-193-mediated trapping as an annulus on closed circular DNA. These data imply that the mutation interferes directly with ICRF-193 binding to the N-terminal ATPase gate. In contrast, the A648P enzyme displayed topoisomerase activities exhibiting wild-type sensitivity to ICRF-193. We suggest that the inefficient trapping of the A648P closed clamp results either from the observed increased ATP requirement, or more likely, from lowered salt stability, perhaps involving destabilization of ICRF-193 interactions with the B'-B' interface in the core domain. These results provide evidence for at least two different phenotypic classes of ICRF-193 resistance mutations and suggest that bisdioxopiperazine action involves the interplay of both the ATPase and core domains of topoisomerase IIalpha.

Adenosine Triphosphatases↗
PubMed1986

Separation of large DNA molecules by contour-clamped homogeneous electric fields.

Electric fields can be manipulated by a method in which multiple electrodes are arranged along a closed contour and clamped to predetermined electric potentials. This method may be applied to a broad range of problems in the separation of macromolecules by gel electrophoresis. DNA molecules as large as 2 megabases can be well separated with a contour-clamped homogeneous electric field alternating between two orientations 120 degrees apart. The pattern of separation is independent of position in the gel, which is an advantage over previous methods. DNA less than 50 kilobases can be separated without distortion even at high voltage with a nonalternating contour-clamped homogeneous field. Decreased band broadening in DNA less than 200 bases can be achieved with a contour-clamped inhomogeneous field.

DNA↗
PubMed1979

Glucose clamp technique: a method for quantifying insulin secretion and resistance.

Methods for the quantification of beta-cell sensitivity to glucose (hyperglycemic clamp technique) and of tissue sensitivity to insulin (euglycemic insulin clamp technique) are described. Hyperglycemic clamp technique. The plasma glucose concentration is acutely raised to 125 mg/dl above basal levels by a priming infusion of glucose. The desired hyperglycemic plateau is subsequently maintained by adjustment of a variable glucose infusion, based on the negative feedback principle. Because the plasma glucose concentration is held constant, the glucose infusion rate is an index of glucose metabolism. Under these conditions of constant hyperglycemia, the plasma insulin response is biphasic with an early burst of insulin release during the first 6 min followed by a gradually progressive increase in plasma insulin concentration. Euglycemic insulin clamp technique. The plasma insulin concentration is acutely raised and maintained at approximately 100 muU/ml by a prime-continuous infusion of insulin. The plasma glucose concentration is held constant at basal levels by a variable glucose infusion using the negative feedback principle. Under these steady-state conditions of euglycemia, the glucose infusion rate equals glucose uptake by all the tissues in the body and is therefore a measure of tissue sensitivity to exogenous insulin.

Glucose↗
PubMed1987

The pancreatic-adrenocortical-pituitary clamp technique for study of counterregulation in humans.

The present experiments were undertaken to develop an approach to analyze the contribution of individual glucose counterregulatory hormones in humans. For this purpose, 24 normal subjects were studied twice: once (control experiments) hypoglycemia was induced by subcutaneous infusion of insulin; and once [pancreatic-adrenocortical-pituitary (PAP) clamp technique] the spontaneous responses of plasma glucagon, growth hormone, and cortisol to hypoglycemia were prevented by intravenous somatostatin and oral metyrapone, respectively, and each hormone was infused at variable rates, which reproduced spontaneous changes in their circulating concentrations in the control experiments. Plasma glucose rate of decrease (0.052 +/- 0.003 vs. 0.06 +/- 0.003 mg X dl-1 X min-1), plasma glucose nadir (49.8 +/- 1.2 vs. 50 +/- 1.0 mg/dl), initial suppression of glucose production (0.22 +/- 0.01 vs. 0.23 +/- 0.01 mg X kg-1 X min-1), subsequent compensatory increase in glucose production (0.54 +/- 0.05 vs. 0.48 +/- 0.04 mg X kg-1 X min-1), and the increase in glucose utilization (0.45 +/- 0.05 vs. 0.42 +/- 0.05 mg X kg-1 X min-1) in PAP clamp and control experiments, respectively, were not significantly different and were significantly correlated. Changes in plasma alanine, lactate, free fatty acids, 3-beta-hydroxybutyrate concentrations were also virtually identical in the PAP clamp experiments and in control experiments. We conclude that the PAP clamp technique can faithfully reproduce the spontaneous hormonal and substrate responses to hypoglycemia and should be useful to assess the contribution of individual hormones during counterregulation by creating an isolated (total or partial) deficiency of a particular hormone without confounding compensatory changes in secretion of other counterregulatory hormones.

3-Hydroxybutyric Acid↗
PubMed1997

Serum free IGF-I during a hyperinsulinemic clamp following 3 days of administration of IGF-I vs. saline.

In a randomized crossover study in eight healthy subjects, we compared the effect of 3 days of continuous subcutaneous administration of insulin-like growth factor I (IGF-I; 10 micrograms.kg-1.h-1) and saline on fasting serum levels of free IGF-I, total (extractable) IGF-I, and IGF-binding protein (IGFBP)-1 and -3. On the 3rd day a hyperinsulinemic (euglycemic and hypoglycemic) clamp was performed. When preclamp (baseline) levels were compared after 3 days, IGF-I administration had increased total IGF-I from 225 +/- 21 (means +/- SE) to 1,003 +/- 46 micrograms/l (P < 0.0001), free IGF-I from 0.5 +/- 0.2 to 10.4 +/- 1.7 micrograms/l (P < 0.001), IGFBP-3 from 2,908 +/- 148 to 3,591 +/- 179 micrograms/l (P < 0.01), and IGFBP-1 from 7.6 +/- 3.8 to 19.6 +/- 2.5 micrograms/l (P < 0.01). During the clamp, levels of free IGF-I increased gradually from baseline to 1.0 +/- 0.3 micrograms/l (saline; P < 0.01) and to 19.6 +/- 4.7 micrograms/l (IGF-I; P < 0.005). Concomitantly, levels of IGFBP-1 decreased gradually from baseline to 4.1 +/- 2.3 micrograms/l (saline; P < 0.0005) and to 4.6 +/- 1.8 micrograms/l (IGF-I; P < 0.0001). Total IGF-I exhibited minor changes only during the clamp (P < 0.05), and IGFBP-3 was unchanged. In conclusion, administration of IGF-I increased total IGF-I about fourfold, whereas free IGF-I increased 20-fold. Noteworthily, in both situations a further twofold increase in free IGF-I was observed during the hyperinsulinemic clamp, concomitant with a decrease in IGFBP-1. This supports the hypothesis that IGFBP-1 is important in the short-term regulation of free IGF-I in vivo.

Adult↗
PubMed1993

Effect of inhibitors of gastric secretion on polarization of gastric potential by voltage clamping.

Voltage clamping across the frog gastric mucosa, nutrient positive to secretory, results in an increase in the open-circuit potential difference (OCPD). The increase in OCPD, or polarization, induced by voltage clamping could be caused by a redistribution of ions across the plasma membrane or by a polarization of pump electromotive forces, such as the Na(+)-K(+)-adenosinetriphosphatase (ATPase) located in the nutrient (serosal) membrane or the proton pump located in the secretory (luminal) membrane. The polarization induced by voltage clamping was not affected by removing Cl- and Na+ or by increasing the K+ concentration to 79 mM but was markedly reduced by placing in the nutrient solution 10(-3) M famotidine or 10(-4) M omeprazole. These data suggest that there is a strong contribution of the proton pump to the polarization induced by voltage clamping with some contribution of ion redistribution and/or the Na(+)-K(+)-ATPase. The data support the electrogenicity of the proton pump.

Animals↗
PubMed1990

Angiotensin and alpha-adrenoceptor activation play a role in hemodynamic response to aortic cross-clamping.

This study was designed to identify the possible role of vasoconstricting compounds released from ischemic tissues in the hemodynamic response to cross-clamping of the thoracic aorta. Twenty-one dogs were anesthetized with pentobarbital sodium. The left hindlimb was denervated, vascularly isolated, and pump perfused at a constant rate with blood drained from the inferior vena cava after passing through a gas-exchanging membrane where oxygen and carbon dioxide tensions were maintained within normal limits. Left and right thoracotomies were performed, and the aorta and inferior vena cava were cross-clamped. The cross-clamping was associated with 33-45% increase in limb vascular resistance in denervated control animals (n = 6). In animals pretreated with Enalaprilat (2 mg/kg, n = 6), an angiotensin-converting enzyme inhibitor, limb vascular resistance did not change significantly. In animals pretreated with phenoxybenzamine (3 mg/kg, n = 6), an alpha-adrenoceptor antagonist, limb vascular resistance significantly decreased to 43% of preclamped level. The study demonstrated that vasoconstrictive compounds, such as angiotensin and catecholamines, play a role in systemic hemodynamic changes, including arterial hypertension, observed during cross-clamping of the thoracic aorta.

Angiotensin II↗
PubMed2004

Predominant postglomerular vascular resistance response to reflex renal sympathetic nerve activation during ANG II clamp in rabbits.

We have shown previously that a moderate reflex increase in renal sympathetic nerve activity (RSNA) elevated glomerular capillary pressure, whereas a more severe increase in RSNA decreased glomerular capillary pressure. This suggested that the nerves innervating the glomerular afferent and efferent arterioles could be selectively activated, allowing differential control of glomerular capillary pressure. A caveat to this conclusion was that intrarenal actions of neurally stimulated ANG II might have contributed to the increase in postglomerular resistance. This has now been investigated. Anesthetized rabbits were prepared for renal micropuncture and RSNA recording. One group (ANG II clamp) received an infusion of an angiotensin-converting enzyme inhibitor (enalaprilat, 2 mg/kg bolus plus 2 mg.kg(-1).h(-1)) plus ANG II ( approximately 20 ng.kg(-1).min(-1)), the other vehicle. Measurements were made before (room air) and during 14% O(2). Renal blood flow decreased less during ANG II clamp compared with vehicle [9 +/- 1% vs. 20 +/- 4%, interaction term (P(GT)) < 0.05], despite a similar increase in RSNA in response to 14% O(2) in the two groups. Arterial pressure and glomerular filtration rate were unaffected by 14% O(2) in both groups. Glomerular capillary pressure increased from 33 +/- 1 to 37 +/- 1 mmHg during ANG II clamp and from 33 +/- 2 to 35 +/- 1 mmHg in the vehicle group before and during 14% O(2), respectively (P(GT) < 0.05). During ANG II clamp, postglomerular vascular resistance was still increased in response to RSNA during 14% O(2), demonstrating that the action of the renal nerves on the postglomerular vasculature was independent of the renin-angiotensin system. This further supports our hypothesis that increases in RSNA can selectively control pre- and postglomerular vascular resistance and therefore glomerular ultrafiltration.

Angiotensin II↗
PubMed1986

Currents under voltage clamp of burst-forming neurons of the cardiac ganglion of the lobster (Homarus americanus).

Crustacean cardiac ganglion neuronal somata, although incapable of generating action potentials, produce regenerative, slow (greater than 200 ms) depolarizing potentials reaching -20 mV (from -50 mV) in response to depolarizing stimuli. These potentials initiate a burst of action potentials in the axon and are thus termed driver potentials. The somata of the anterior-most neurons (cells 1 or 2) were isolated by ligaturing for study of their membrane currents with a two-electrode voltage clamp. Inward current is attributed to Ca2+ by reason of dependence of driver potential amplitude on [Ca2+]0, independence of [Na+]0, resistance to tetrodotoxin, and inhibition by Cd (0.2 mM) and Mn (4 mM). Ca-mediated current (ICa) is present at -40 mV. It is optimally activated by a holding potential (Vh) of -50 to -60 mV and by clamps (command potential, Vc) to -10 mV. Time to peak (10-30 ms) and amplitude are strongly voltage dependent. Maximum tail-current amplitudes observed at -70 to -85 mV are ca. 100 nA. Inward tail peaks may not be resolved by our clamp (settling time, 2 ms). Tails relax with a time constant (tau) of approximately equal to 12 ms (at -70 to -85 mV). ICa exhibits inactivation in double pulse regimes. Recovery has a tau of approximately equal to 0.7 s. Tail current analyses indicate an exponential decline (tau approximately equal to 23 ms at -20 mV) toward a maintained amplitude of inward current tails. Analysis of outward currents indicates the presence of three conductance mechanisms having voltage dependences, time courses, and pharmacology similar to those of early outward current (IA), delayed outward current (IK), and outward current (IC) of molluscan neurons. Analysis of tail currents indicates a reversal potential for each of these near -75 mV, indicating that they are K currents. Early outward current, IA, shows a peak at 5 ms followed by rapid decline. Response to a second clamp given within 0.4 s is reduced; recovery is exponential, with a tau of approximately equal to 200 ms (at Vh = -50 mV). The amplitude of IA tested at 0 mV shows activation or deactivation by subthreshold shifts of Vh. The extent and rate of these changes shows voltage dependence (tau approximately equal to 100-500 ms for subthreshold prepulses). At the normal cell resting potential of -50 mV the amplitude of IA is 25% of that tested from -80 mV.(ABSTRACT TRUNCATED AT 400 WORDS)

4-Aminopyridine↗
PubMed1991

A model of a CA3 hippocampal pyramidal neuron incorporating voltage-clamp data on intrinsic conductances.

1. We have developed a 19-compartment cable model of a guinea pig CA3 pyramidal neuron. Each compartment is allowed to contain six active ionic conductances: gNa, gCa, gK(DR) (where DR stands for delayed rectifier), gK(A), gK(AHP), and gK(C). THe conductance gCa is of the high-voltage activated type. The model kinetics for the first five of these conductances incorporate voltage-clamp data obtained from isolated hippocampal pyramidal neurons. The kinetics of gK(C) are based on data from bullfrog sympathetic neurons. The time constant for decay of submembrane calcium derives from optical imaging of Ca signals in Purkinje cell dendrites. 2. To construct the model from available voltage-clamp data, we first reproduced current-clamp records from a model isolated neuron (soma plus proximal dendrites). We next assumed that ionic channel kinetics in the dendrites were the same as in the soma. In accord with dendritic recordings and calcium-imaging data, we also assumed that significant gCa occurs in dendrites. We then attached sections of basilar and apical dendritic cable. By trial and error, we found a distribution (not necessarily unique) of ionic conductance densities that was consistent with current-clamp records from the soma and dendrites of whole neurons and from isolated apical dendrites. 3. The resulting model reproduces the Ca(2+)-dependent spike depolarizing afterpotential (DAP) recorded after a stimulus subthreshold for burst elicitation. 4. The model also reproduces the behavior of CA3 pyramidal neurons injected with increasing somatic depolarizing currents: low-frequency (0.3-1.0 Hz) rhythmic bursting for small currents, with burst frequency increasing with current magnitude; then more irregular bursts followed by afterhyperpolarizations (AHPs) interspersed with brief bursts without AHPs; and finally, rhythmic action potentials without bursts. 5. The model predicts the existence of still another firing pattern during tonic depolarizing dendritic stimulation: brief bursts at less than 1 to approximately 12 Hz, a pattern not observed during somatic stimulation. These bursts correspond to rhythmic dendritic calcium spikes. 6. The model CA3 pyramidal neuron can be made to resemble functionally a CA1 pyramidal neuron by increasing gK(DR) and decreasing dendritic gCa and gK(C). Specifically, after these alterations, tonic depolarization of the soma leads to adapting repetitive firing, whereas stimulation of the distal dendrites leads to bursting. 7. A critical set of parameters concerns the regulation of the pool of intracellular [Ca2+] that interacts with membrane channels (gK(C) and gK(AHP)), particularly in the dendrites.(ABSTRACT TRUNCATED AT 400 WORDS)

Animals↗
PubMed1993

Effects of soma isolation on outward currents measured under voltage clamp in spiny lobster stomatogastric motor neurons.

1. Outward currents in identified cell types from the pyloric system of the stomatogastric ganglion (STG) of the spiny lobster, Panulirus marginatus, were studied under two-microelectrode voltage clamp. A comparison was made between data from intact cells and somata isolated by ligation of the primary neurite of these monopolar neurons. 2. Despite the elimination of current contributions from the extensive arborizations of STG neurons, few significant differences were found in the mean values of parameters for outward currents between populations of isolated somata and intact cells of a given type. Measurements that showed little difference included magnitude and activation threshold of a calcium-dependent outward current (IJ) and magnitude, activation threshold, voltage dependence, and inactivation time course of A current (IA). Although previous work has suggested that IJ might reside predominantly in the soma, IA is known to be distributed in poorly space-clamped neurite processes. The absence of obvious effects of isolation was thus unexpected. 3. To better understand the mechanisms involved, we used compartmental models derived from reconstructed neurons to simulate the effects of isolation. It was concluded that, for the particular conditions present in stomatogastric neurons, with a large, uniformly distributed outward current conductance activated, even though neurites and axon remain attached, most measured current flows through well-clamped soma membrane. 4. Factors contributing to this result included the outward sign of the current, the large specific conductance activated in these neurons (among the larger reported in somata), and the presence of only a single major process leaving the soma. The potential for serious errors in voltage-clamp measurements from intact cells remains if these conditions are not met.

Animals↗