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PubMed1992

Synergistic effect of light and fusicoccin on stomatal opening : epidermal peel and patch clamp experiments.

Upon incubation of epidermal peels of Commelina communis in 1 millimolar KCl, a synergistic effect of light and low fusicoccin (FC) concentrations on stomatal opening is observed. In 1 millimolar KCl, stomata remain closed even in the light. However, addition of 0.1 micromolar FC results in opening up to 12 micrometers. The same FC concentration stimulates less than 5 micrometers of opening in darkness. The synergistic effect (a) decreases with increasing FC or KCl concentrations; (b) is dark-reversible; (c) like stomatal opening in high KCl concentrations (120 millimolar) is partially inhibited by the K(+) channel blocker, tetraethyl-ammonium(+) (20 millimolar). In whole-cell patch-clamp experiments with guard cell protoplasts of Vicia faba, FC (1 or 10 micromolar) stimulates an increase in outward current that is essentially voltage independent between - 100 and +60 millivolts, and occurs even when the membrane potential is held at a voltage (-60 millivolts) at which K(+) channels are inactivated. These results are indicative of FC activation of a H(+) pump. FC effects on the magnitude of inward and outward K(+) currents are not observed. Epidermal peel and patch clamp data are both consistent with the hypothesis that the plasma membrane H(+) ATPase of guard cells is a primary locus for the FC effect on stomatal apertures.

Journal Article↗
PubMed2006

The Clp1 protein is required for clamp formation and pathogenic development of Ustilago maydis.

In the phytopathogenic fungus Ustilago maydis, pathogenic development is controlled by a heterodimer of the two homeodomain proteins bE and bW, encoded by the b-mating-type locus. We have identified a b-dependently induced gene, clampless1 (clp1), that is required for the proliferation of dikaryotic filaments in planta. We show that U. maydis hyphae develop structures functionally equivalent to clamp cells that participate in the distribution of nuclei during cell division. In clp1 mutant strains, dikaryotic filaments penetrate the plant cuticle, but development is stalled before the first mitotic division, and the clamp-like structures are not formed. Although clp1 is immediately activated upon b-induction on the transcriptional level, nuclear-localized Clp1 protein is first observed at the stage of plant penetration prior to the first cell division. Induced expression of clp1 strongly interferes with b-dependent gene regulation and blocks b-dependent filament formation and b-dependent cell cycle arrest. We speculate that the Clp1 protein inhibits the activity of the bE/bW heterodimer to facilitate the cell cycle progression during hyphal growth.

Basidiomycota↗
PubMed1980

Renin uptake by the right kidney in the rat following partial clamping of the left renal artery.

Renin secretion from both kidneys was studied in eleven Wistar rats for 40 min after partial clamping of the left renal artery. 14C Inulin and 3H para aminohippuric acid clearance and the arterial and venous plasma renin concentrations were measured in each kidney during control and experimental procedures. After clamping, the arterial plasma renin concentration increased and correlated positively with the plasma renin concentration in both renal veins. However, the plasma renin concentration of the right renal vein was lower than the arterial concentration (P < 0.01). The slope correlating the two variables is significantly lower than unity (P < 0.05, n = 19). As the left kidney secreted a large amount of renin, calculation of secretion by the right kidney decreased to negative values, implying renin uptake by this kidney. These data suggest the existence of a balance between the two kidneys. Renin secretion appears to maintain an equilibrium between release by the juxtaglomerular apparatus and removal from the blood into interstitial space. Release seems to predominate in the hypoperfused kidney, and removal in the contralateral kidney. This may result from the increase of the circulating angiotensin II, since we observed a direct correlation between renin uptake and the arterial plasma renin concentration.

Animals↗
PubMed1990

Evaluation of a euglycaemic clamp procedure as a diagnostic test in insulinoma patients.

In 15 patients with insulinoma, six patients after successful removal of this tumour, two patients with previous pancreas resection because of hypoglycaemia elsewhere, and 10 control subjects, the diagnostic usefulness of euglycaemic clamp procedures (without exogenous insulin) was assessed in comparison with prolonged starvation. Only insulinoma patients developed sustained hypoglycaemia (less than or equal to 2.3 mmol l-1) within 2-44 h without caloric intake, because of inappropriately elevated immunoreactive insulin (IR-insulin) concentrations. IR-proinsulin values were elevated in most (7 out of 10), but not in all insulinoma patients. The steady-state glucose infusion rate necessary to maintain a stable plasma glucose concentration of 4.4-5.0 mmol l-1 was significantly (P less than or equal to 0.001) higher in insulinoma patients (2.5 +/- 0.6 mg kg-1 min-1) than in pancreas resected patients (0.6 +/- 0.2 mg kg-1 min-1), or in control subjects (0.5 +/- 0.1 mg kg-1 min-1). Due to a considerable degree of overlap, sensitivity (0.44) and specificity (0.95) were too low for such a procedure to qualify as a diagnostic test. There was no correlation of glucose infusion rates to IR-insulin values (r = 0.024, P = 0.461). One reason for this was the development of insulin resistance in some, but not in all insulinoma patients. When, in analogy to insulin/glucose ratios, a diagnostic index was derived by multiplying the steady state glucose infusion rate by the steady state IR-insulin concentration, the diagnostic accuracy was greatly increased (sensitivity and specificity 0.94, respectively), but still lower than that of 'amended' insulin/glucose ratios in fasting plasma or at the time of discontinuation of prolonged fasts (1.00). Somatostatin infusions inhibited insulin secretion (IR-C-peptide plasma concentrations) by 52-88% in subjects without insulinoma and in those insulinoma patients whose tumour cells ultrastructurally contained plenty of normal secretory granules, and to a lesser degree when only abnormal or virtually no secretory granules were present, i.e. in more de-differentiated tumours. In contrast to this significant (P = 0.036) association, malignancy, i.e. the presence of metastases, could not be predicted from whether or not insulin secretion was resistant to the inhibitory action of somatostatin. In conclusion, euglycaemic clamp experiments are less reliable for detecting or excluding a functioning insulinoma than the relation of glucose and insulin values during starvation. The inhibition of insulin secretion by somatostatin depends on the presence of normal beta-granules, and does not distinguish adenomas from carcinomas.

Adenoma, Islet Cell↗
PubMed1992

Increase of glomerular filtration rate and renal plasma flow by insulin-like growth factor-I during euglycaemic clamping in anaesthetized rats.

Renal function was studied in anaesthetized rats receiving i.v. infusions of recombinant human insulin-like growth factor-I (IGF-I) under euglycaemic clamp conditions. IGF-I increased glomerular filtration rate up to 35% and renal plasma flow up to 100%, this increase being dose dependent with half-maximal stimulation at serum IGF-I concentrations of about 24 pmol ml-1. Renal vascular resistance was reduced up to 50%, filtration fraction decreased up to 30% and urine flow increased up to three fold while arterial blood pressure was unchanged. Renal haemodynamics were affected at serum IGF-I concentrations that did not stimulate total body glucose disposal during euglycaemic clamping. IGF-I seemed to regulate renal function through IGF-I receptors apparently independent of acute changes of glucose metabolism.

Animals↗
PubMed2006

Effects of music on patients undergoing a C-clamp procedure after percutaneous coronary interventions.

AIM: This paper reports a study to determine the effect of music on physiological parameters and level of pain in patients undergoing application of a C-clamp after percutaneous coronary interventions. BACKGROUND: Most percutaneous coronary interventions are performed through the femoral artery. In order to stop bleeding and achieve homeostasis, a C-clamp is used after percutaneous coronary interventions. However, the experience is painful for patients and they inevitably suffer discomfort. Pain may lead to stress responses and may affect the physical and mental health of patients. One potential beneficial practice is having the patient listen to relaxing music, which might have the effect of reducing situational discomfort and pain. METHODS: A randomized controlled study was conducted during the period September 2004 to March 2005. Forty-three people (20 experimental and 23 control) were recruited from the intensive care units of two acute care hospitals in Hong Kong. Physiological and psychological variables were collected at baseline and at 15, 30 and 45 minutes. RESULTS: In the music group, there were statistically significant reductions (P=0.001) in heart rate, respiratory rate, and oxygen saturation than the control participants at 45 minutes. In the music group, statistically significant reductions (P=0.001) in systolic blood pressure, heart rate, respiratory rate and oxygen saturation were found at the four time points, but not in the control group. No statistically significant differences were found at baseline comparison of the two groups, but statistically significant differences in pain scores were found at 45 minutes for participants in the music group compared with the control group (P=0.003). Participants in the control group showed statistically significant increases in pain at 45 minutes compared with baseline (P<0.001). CONCLUSIONS: The benefits of preventing physiological reactions to pain were demonstrated. Music is a simple, safe and effective method of reducing potentially harmful physiological and psychological responses arising from pain.

Adult↗
PubMed2006

Two processivity clamp interactions differentially alter the dual activities of UmuC.

DNA polymerases of the Y family promote survival by their ability to synthesize past lesions in the DNA template. One Escherichia coli member of this family, DNA pol V (UmuC), which is primarily responsible for UV-induced and chemically induced mutagenesis, possesses a canonical beta processivity clamp-binding motif. A detailed analysis of this motif in DNA pol V (UmuC) showed that mutation of only two residues in UmuC is sufficient to result in a loss of UV-induced mutagenesis. Increased levels of wild-type beta can partially rescue this loss of mutagenesis. Alterations in this motif of UmuC also cause loss of the cold-sensitive and beta-dependent synthetic lethal phenotypes associated with increased levels of UmuD and UmuC that are thought to represent an exaggeration of a DNA damage checkpoint. By designing compensatory mutations in the cleft between domains II and III in beta, we restored UV-induced mutagenesis by a UmuC beta-binding motif variant. A recent co-crystal structure of the 'little finger' domain of E. coli pol IV (DinB) with beta suggests that, in addition to the canonical beta-binding motif, a second site of pol IV ((303)VWP(305)) interacts with beta at the outer rim of the dimer interface. Mutational analysis of the corresponding motif in UmuC showed that it is dispensable for induced mutagenesis, but that alterations in this motif result in loss of the cold-sensitive phenotype. These two beta interaction sites of UmuC affect the dual functions of UmuC differentially and indicate subtle and sophisticated polymerase management by the beta clamp.

Amino Acid Sequence↗
PubMed1997

Low cardiac output abolishes cardiovascular responses to infra-renal aortic cross-clamping in the pig.

BACKGROUND: Previous data suggest that preoperative myocardial dysfunction is associated with an altered cardiac response to infra-renal aortic cross-clamping (AXC). This study was designed to further explore how acute reductions in stroke volume and cardiac output influence the systemic, preportal and renal circulatory responses to AXC. METHODS: In chloralose-anesthetized normoventilated pigs, graded increases in pericardial pressure (PPERICARD) were obtained by local infusions of dextran. Measurements included cardiac output (CO, thermodilution), mean blood pressure proximal to the aortic clamping site (MAPPROX) and ultrasonic flowmetry for portal (QPORT) and renal (QREN) blood flows. In all animals, measurements were made a) prior to AXC, b) at the end of a 5 min AXC period and, c) 5 min following declamping. These recordings were repeated during control (PPERICARD 0 cmH2O) and during stages with increased PPERICARD (4 and 8 cmH2O, respectively). RESULTS: Pericardial infusions of dextran produced hemodynamic responses that in magnitude were proportional to PPERICARD levels. Stroke volume, CO and mean arterial pressure decreased, while systemic vascular resistance (SVR) increased. In the preportal tissues, vascular resistance increased and QPORT decreased. Similarly, in the kidney, vascular resistance and QREN decreased, but only at a PPERICARD of 8 cmH2O. At control, AXC increased SVR, MAPPROX, QPORT and both renal and preportal vascular resistances. When PPERICARD was increased to 4 cm H2O, the responses to AXC concerning SVR and MAPPROX were not significantly altered, while renal and preportal circulatory responses were blunted. At stages with a PPERICARD of 8 cmH2O, we could not demonstrate any circulatory responses to AXC. CONCLUSIONS: AXC-induced systemic, preportal and renal circulatory responses are inhibited during a condition of acutely lowered cardiac output.

Animals↗
PubMed1990

A delta pH clamp method for analysis of steady-state kinetics of photophosphorylation.

An instrumental device is described which allows steady-state kinetic measurements of photophosphorylation at a desired proton gradient which can be maintained throughout the course of the experiment ('delta pH clamp'). This is achieved by electronic regulation of light intensity using the calibrated 9-aminoacridine fluorescence signal as sensor of the gradient. The instrument is suitable for determination of kinetic parameters of the proton-translocating ATPase in isolated envelope-free chloroplasts under defined conditions. At clamped delta pH, phosphorylation as a function of substrate concentration shows Michaelis-Menten kinetics. The true Michaelis constants and the dissociation constants for phosphate and ADP are reported. The Michaelis constants are not affected by the magnitude of the proton gradient in the investigated range. The significance of these results is discussed.

Aminacrine↗
PubMed2000

Ischaemic changes in refractoriness of human cutaneous afferents under threshold-clamp conditions.

1. A technique was developed to counteract the changes in threshold to electrical stimuli of large myelinated cutaneous afferents in the human median nerve induced by ischaemia for 13 min. Intermittent application of polarizing currents was used in five subjects, in whom refractoriness, supernormality and the strength-duration time constant (tauSD) were tracked to determine whether compensating for the ischaemia-induced changes in threshold also controlled the ischaemic changes in these excitability parameters. 2. The threshold compensation prevented the ischaemic changes in tauSD, an excitability parameter dependent on nodal Na+ channels. Threshold compensation did not prevent the changes in refractoriness and supernormality, whether the compensation began 10, 100 or 200 ms prior to the test stimuli. 3. In three subjects, continuous polarizing current was injected for 13 min to compensate for the ischaemic change in threshold, thus clamping threshold at the pre-ischaemic level. Again, tauSD was effectively controlled, but there were still ischaemic changes in refractoriness and supernormality. 4. The effective control of tauSD suggests that both the intermittent threshold compensation and the continuous threshold clamp effectively controlled membrane potential at the node of Ranvier. 5. The ischaemic increase in refractoriness when threshold was kept constant could be due to interference with the processes responsible for refractoriness by a metabolic product of ischaemia. The ischaemic change in supernormality during effective compensation probably results from the intrusion of refractoriness into the conditioning-test intervals normally associated with maximal supernormality. 6. The present results indicate that ischaemia has effects on axonal excitability that cannot be readily explained by changes in membrane potential. Specifically, it is suggested that ischaemic metabolites interfere with the recovery of Na+ channels from inactivation.

Adaptation, Physiological↗
PubMed1987

Insulin sensitivity in patients with arterial insufficiency. Re-evaluation with the multilevel glucose clamp technique.

Previous studies have claimed that changes in insulin and glucose metabolism in patients with peripheral arterial insufficiency indicate an increased sensitivity to insulin for glucose uptake. We have re-evaluated this concept in 11 patients with intermittent claudication and 11 matched controls with a multilevel euglycaemic glucose clamp technique. The groups were matched for age, sex and body composition but not completely for smoking habits and physical fitness. Although the control group had a normal physical fitness the patients had 40% lower maximal exercising capacity. The patients' maximal walking capacity was 186 +/- 27 m evaluated on a treadmill. No patient with ischaemic ulcers or rest pain were included. The euglycaemic glucose clamp was performed at five insulin plateau levels (70, 110, 190, 590 and 1440 mU/l) of one h duration each and whole body glucose uptake during the last 20 min was calculated. No differences in maximal glucose uptake (Vmax) or insulin level for half maximal glucose uptake (Km) between the groups were observed. The concentration or the magnitude of the decrease in arterial FFA levels did not differ significantly between the groups at any physiological insulin level. This study can not confirm previously described changes in insulin sensitivity in patients with arterial insufficiency. Although the discrepancy between present and previous results remains unclear it may reflect the combined effects of different methods used, including the selection of reference patients, rather than an increased insulin sensitivity in patients with arterial insufficiency. This study suggests that previous conclusions based on glucose tolerance tests should be interpreted with caution.

Biological Transport, Active↗
PubMed1996

Comparison of hyperinsulinaemic clamp experiments using venous, 'arterialized' venous or capillary euglycaemia.

It has been suggested that deviations from arterial euglycaemia during hyperinsulinaemic clamp experiments that use venous plasma glucose measurements may alter the results of such tests (glucose infusion rate, C-peptide suppression, etc.) and that 'arterialized' venous blood ('heated-hand' technique) may be suitable to circumvent these problems. Therefore, nine normal male fasting volunteers (age 25 +/- 4 years, body mass index 23.5 +/- 2.3 kg m-2) were examined three times using an insulin infusion of 1 mU kg min-1 over 120 min. Glucose was infused to maintain a concentration of 4.7 mmol I-1 (85 mg dl-1) in venous (V), 'arterialized' venous ('heated-hand' technique; HH), or capillary (C) plasma. The 'heated-hand' technique caused a rise in (rectal) body temperature of 0.3 +/- 0.1 degree C (P < 0.0001). Whereas the glucose aim was reached to a similar degree in all experiments (P = 0.36), capillary glucose concentrations differed slightly, but significantly (higher in experiments with venous and 'arterialized' venous blood specimens; P = 0.034). There were no significant differences regarding steady-state insulin concentrations (P = 0.77), glucose infusion rates (V, 7.1 +/- 0.5; HH, 7.2 +/- 0.6; C, 6.4 +/- 0.5 mg kg-1 min-1; P = 0.98), C-peptide suppression (P = 0.78), reduction in glucagon (P = 0.27) and free fatty acids (P = 0.16), and all parameters of indirect calorimetry (non-protein RQ: P = 0.67; glucose and lipid oxidation: P = 0.72 and 0.46 respectively; and energy expenditure: P = 0.42). Therefore, hyperinsulinaemic clamp experiments performed using venous, 'arterialized' venous, or capillary euglycaemia appear to be almost equally useful for the determination of insulin sensitivity and C-peptide or glucagon suppression. The elevation in body temperature that accompanies use of the 'heated-hand' technique does not noticeably influence measured metabolic parameters.

Adult↗
PubMed1976

The effect of quinidine on membrane electrical activity in frog auricular fibres studied by current and voltage clamp.

1 The action of quinidine sulphate 50 muM has been investigated on frog auricular trabeculae transmembrane currents recorded with a double sucrose gap apparatus. Results were obtained either in current or in voltage clamp conditions. 2 Quinidine modified the time course of repetitive activity elicited by long lasting depolarizing currents and reduced the range of current over which repetitive activity could be triggered, eventually abolishing repetitive responses altogether. 3 Several authors have emphasized the limitations of the voltage clamp method. Taking into account these limitations, the numerical values of the parameters obtained in the present work must not be considered as exact values but may be interpreted as indicators of the variations of the parameters. 4 The results are in agreement with previous findings that the main features of the action of quinidine are to produce (a) a reduced maximum rate of depolarization (MRD), (b) a reduced total amplitude of action potential, (c) a flattening of the plateau of the action potential, (d) a slight prolongation of the tail of the action potential, (e) an increased effective refractory period without greatly prolonging action potential duration, (f) no change of resting potential and of 50% repolarization time. 5 The analysis of ionic conductances has provided explanations for the above effects. 6 Quinidine reduced the reactivation kinetics of the sodium inward current, and decreased sodium conductance and the steady state of activation. These effects account for (a) and (b). 7 Quinidine increased the activation and inactivation time constants of sodium conductances, which account in part for (e). 8 Quinidine delayed reactivation of slow inward current, reduced calcium conductance, and decreased the steady state of activation of calcium conductance. These effects could account for (c). 9 The amplitudes of the two components of the delayed conductances responsible for repolarization were decreased by quinidine, and the time constant of activation for the faster of the two was slowed. These effects could account for (d) and in part for (e).

Action Potentials↗
PubMed1984

Alteration of the fast excitatory postsynaptic current by barium in voltage-clamped amphibian sympathetic ganglion cells.

Barium-induced alterations in fast excitatory postsynaptic currents (e.p.s.cs) have been studied in voltage-clamped bullfrog sympathetic ganglion B cells. In the presence of 2-8 mM barium, e.p.s.c. decay was prolonged and in many cells the e.p.s.c. decay phase deviated from a single exponential function. The decay phase in these cases was more accurately described as the sum of two exponential functions. The frequency of occurrence of a complex decay increased both with increasing barium concentration and with hyperpolarization. Miniature e.p.s.c. decay also was prolonged in barium-treated cells. E.p.s.c. amplitude was not markedly affected by barium (2-8 mM) in cells voltage-clamped to -50 mV whereas at -90 mV there was a progressive increase in peak size with increasing barium concentration. In control cells the e.p.s.c.-voltage relationship was linear between -20 and -100 mV; however, this relationship became progressively non-linear with membrane hyperpolarization in barium-treated cells. The e.p.s.c. reversal potential was shifted to a more negative value in the presence of barium. There was a voltage-dependent increase in charge movement during the e.p.s.c. in barium-treated cells which was not present in control cells. We conclude that the voltage-dependent alteration in e.p.s.c. decay time course, peak amplitude and charge movement in barium-treated cells is due to a direct postsynaptic action of barium on the kinetics of receptor-channel gating in postganglionic sympathetic neurones.

Action Potentials↗
PubMed1988

Interactions between molecules of a steroid anaesthetic (alphaxalone) and ionic channels of nodal membrane in voltage-clamped myelinated nerve fibre.

1. The effects of the anaesthetic alphaxalone (0.05 to 1 mM) on the node of Ranvier of isolated myelinated nerve fibres of the frog were studied under voltage-clamp conditions. 2. When added to the solution bathing voltage-clamped nodes, alphaxalone modified neither linear leakage nor capacitative currents but rapidly and reversibly blocked K and Na currents. The blocking effects of the anaesthetic on both types of current were not dependent on the frequency of stimulation of the nerve fibres between 0.7 and 10 Hz. 3. The kinetics of the Na current were not modified by alphaxalone but, in the presence of the drug, the K current showed an apparent fast inactivation. 4. Alphaxalone rapidly and reversibly shifted towards negative voltages both the steady-state K conductance-voltage and the peak Na steady-state inactivation-voltage relationships, without noticeable modification of their shape. In contrast, the anaesthetic reversibly decreased the slope of the peak Na conductance-voltage curve. 5. The reduction of the K current induced by alphaxalone was voltage-dependent with an apparent dissociation constant first decreasing from about 0.25 to 0.08 mM between -20 mV and +20 mV and then remaining constant above +20 mV. In contrast, the apparent dissociation constant for the Na current was almost constant with increasing voltages and equalled about 0.30 mM. Hill coefficient values for both K and Na currents were noticeably less than one. 6. It is concluded that, at higher concentrations than those attainable in the brain or in the plasma during surgical anaesthesia in man, alphaxalone has a 'local anaesthetic-like' action on the peripheral nervous system in that it specifically and differentially interacts with K and Na channel gating systems: it is suggested that the anaesthetic would preferentially modify open K and inactivated Na channels.

Anesthetics↗
PubMed1989

The mechanism of action and pharmacological specificity of the anticonvulsant NMDA antagonist MK-801: a voltage clamp study on neuronal cells in culture.

1. Some possible molecular mechanisms of action of the anxiolytic, anticonvulsant and neuroprotective agent MK-801 have been examined in 'whole-cell' voltage clamp recordings performed on rat hippocampal and cortical neurones, bovine adrenomedullary chromaffin cells and N1E-115 neuroblastoma cells maintained in cell culture. 2. Transmembrane currents recorded from rat hippocampal and cortical neurones in response to locally applied N-methyl-D-aspartate (NMDA) were antagonized by MK-801 (0.1-3.0 microM). Blockade was use-dependent, and little influenced by transmembrane potential. MK-801 (3 microM) had no effect on currents evoked by kainate (100 microM). 3. The antagonism of NMDA-induced currents by MK-801 was only slowly and incompletely reversed when the cell membrane potential was clamped at -60 mV during washout. Prolonged applications of NMDA at +40, but not -60 mV during washout, markedly accelerated recovery from block. 4. In contrast to MK-801, ketamine (10 microM) blocked NMDA-induced currents in a voltage-dependent manner. Blockade increased with membrane hyperpolarization and was completely reversible upon washout. 5. MK-801 (1-10 microM) produced a voltage- and concentration-dependent block of membrane currents elicited by ionophoretically applied acetylcholine (ACh) recorded from bovine chromaffin cells. The block was readily reversible upon washout. 6. gamma-Aminobutyric acidA (GABAA) receptor-mediated chloride currents of chromaffin cells were unaffected by MK-801 (1-100 microM). In contrast, such currents were potentiated by diazepam (1 microM). MK-801 (100 microM) had no effect on currents evoked by GABA on hippocampal neurones. 7. MK-801 (10 microM) had little effect on membrane currents recorded from N1E-115 neuroblastoma cells in response to ionophoretically applied 5-hydroxytryptamine (5-HT). Such currents were antagonized by the 5-HT3 receptor antagonist GR 38032F (1 nM) and also by MK-801 at high concentration (100 microM). 8. Voltage-activated, tetrodotoxin-sensitive, sodium currents of chromaffin cells were unaffected by 10 microM MK-801. However, at a relatively high concentration (100 microM), MK-801 reduced the amplitude of such currents to approximately 77% of control. 9. The relevance of the present results to the central actions of MK-801 is discussed.

Acetylcholine↗
PubMed1989

The properties of 5-HT3 receptors in clonal cell lines studied by patch-clamp techniques.

1 The characteristics of transmembrane currents evoked by 5-hydroxytryptamine (5-HT) in the neuroblastoma x Chinese hamster brain cell line NCB-20 and neuroblastoma clonal cell line N1E-115 have been studied under voltage-clamp conditions by the whole-cell recording and outside-out membrane patch modes of the patch-clamp technique. 2 In 73% of NCB-20 cells examined (n = 221), and all N1E-115 cells studied (n = 80), 5-HT (10 microM) elicited a transient inward current at negative holding potentials, this being associated with an increase in membrane conductance. In both cell lines responses to 5-HT reversed in sign at a potential of approximately -2 mV and demonstrated inward rectification. 3 The reversal potential of 5-HT-induced currents (E5-HT) recorded from either NCB-20 or N1E-115 cells was unaffected by total replacement of internal K+ by Cs+. In N1E-115 cells, reducing internal K+ concentration from 140 to 20 mM produced a positive shift in E5-HT of approximately 28 mV, whereas reducing external Na+ from 143 to 20 mM was associated with a negative shift in E5-HT of about 37 mV. A large reduction in internal Cl- concentration (from 144 to 6 mM) had little effect on E5-HT. 4 5-HT-induced currents of NCB-20 cells were unaffected by methysergide (1 microM) or ketanserin (1 microM), but were reversibly antagonized by GR38032F (0.1-1.0 nM) with an IC50 of 0.25 nM. GR 38032F (0.3 nM) reduced 5-HT-induced currents in N1E-115 cells to approximately 26% of their control value. 5 On outside-out membrane patches excised from both NCB-20 and N1E-115 cells, 5-HT induced small inward currents which could not be clearly resolved into discrete single channel events. Such responses were: (i) reversibly antagonized by GR 38032F (1 nM) (ii) reversed in sign at 0 mV, and (iii) subject to desensitization. 6 Fluctuation analysis of inward currents evoked by 5-HT (1 microM) in N1E-115 cells suggests that 5-HT gates a channel with a conductance of approximately 310fS. Such a relatively small conductance could readily explain why the response of outside-out membrane patches to 5-HT cannot at present be resolved into clear single channel events.

Cell Membrane↗
PubMed1989

Actions of guanine nucleotides and cyclic nucleotides on calcium stores in single patch-clamped smooth muscle cells from rabbit portal vein.

1. Single smooth muscle cells were obtained from the rabbit portal vein by enzymic digestion and membrane currents under voltage clamp measured by whole-cell patch clamp technique. 2. When held at depolarized potentials, spontaneous outward currents (STOCs) were discharged; it is likely that these represent the cyclical storage and release within the cell of calcium in relation to Ca-activated K-channels. 3. Application of lower concentrations of carbachol (10(-5)M) or caffeine (10(-3)M) accelerated STOC discharge. Higher concentrations of caffeine (10(-2)M) or carbachol (10(-4)M), or noradrenaline (10(-5)M), produced an outward current of 1-5 nA which disappeared within 5-15s and which was considered to result from the discharge of calcium stores; STOC discharge was abolished for a period. 4. Ryanodine (10(-5)-10(-4)M) or a non-hydrolysable GTP analogue, GTP gamma S (10(-5)-10(-3)M) introduced into the cell abolished STOC discharge within 2-5 min. STOCs were large in cells filled with GDP beta S (10(-3)M) and the action of GTP gamma S introduced at various concentrations was antagonized. 5. GTP gamma S (10(-4)-10(-3)M) in the cell reduced or abolished outward current to caffeine (10(-2)M) noradrenaline (10(-5)M) or carbachol (10(-4)M); the effect on caffeine outward current was antagonized by GDP beta S (10(-3)M) introduced into the cell. GDP beta S reduced noradrenaline outward current but not caffeine outward current implying the existence of a G-protein step in noradrenaline-evoked Ca-store release, possibly regulating phospholipase C enzyme activity and D-myo inositol 1,4,5 trisphosphate formation. 6. If cyclic AMP (10(-3)M) or cyclic GMP (10(-3)M) was introduced into the cell, or 8-bromo cyclic AMP (0.5 x 10(-3)M) or 8-bromo cyclic GMP (0.5 x 10(-3)M) applied to the cell in the bathing solution, STOC discharge was only slightly affected. However, the outward current to caffeine applied after noradrenaline was much enhanced. 7. The results could be explained if cyclic GMP and cyclic AMP enhance calcium storage whereas GTP gamma S depletes calcium stores, an action antagonized by GDP beta S.

8-Bromo Cyclic Adenosine Monophosphate↗