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R E Wachtel

Publications and source records attributed to R E Wachtel.

At least 19 recordsLinked to original sources
PubMed2001

Flow-induced dilation of human coronary arterioles: important role of Ca(2+)-activated K(+) channels.

BACKGROUND: Flow-induced vasodilation (FID) is a physiological mechanism for regulating coronary flow and is mediated largely by nitric oxide (NO) in animals. Because hyperpolarizing mechanisms may play a greater role than NO in the microcirculation, we hypothesized that hyperpolarization contributes importantly to FID of human coronary arterioles. METHODS AND RESULTS: Arterioles from atria or ventricles were cannulated for videomicroscopy. Membrane potential of vascular smooth muscle cells (VSMCs) was measured simultaneously. After constriction with endothelin-1, increases in flow induced an endothelium-dependent vasodilation. Nomega-Nitro-L-arginine methyl ester 10(-4) mol/L modestly impaired FID of arterioles from patients without coronary artery disease (CAD), whereas no inhibition was seen in arterioles from patients with CAD. Indomethacin 10(-5) mol/L was without effect, but 40 mmol/L KCl attenuated maximal FID. Tetraethylammonium 10(-3) mol/L but not glibenclamide 10(-6) mol/L reduced FID. Charybdotoxin 10(-8) mol/L impaired both FID (15+/-3% versus 75+/-12%, P<0.05) and hyperpolarization (-32+/-2 mV [from -28+/-2 mV after endothelin-1] versus -42+/-2 mV [-27+/-2 mV], P<0.05). Miconazole 10(-6) mol/L or 17-octadecynoic acid 10(-5) mol/L reduced FID. By multivariate analysis, age was an independent predictor for the reduced FID. Conclusions-We conclude that shear stress induces endothelium-dependent vasodilation, hyperpolarizing VSMCs through opening Ca(2+)-activated K(+) channels in human coronary arterioles. In subjects without CAD, NO contributes to FID. NO and prostaglandins play no role in patients with CAD; rather, cytochrome P450 metabolites are involved. This is consistent with a role for endothelium-derived hyperpolarizing factor in FID of the human coronary microcirculation.

Age Factors↗
PubMed2000

Mechanosensitive potassium channels in rat colon sensory neurons.

Single-channel recording techniques were used to characterize mechanosensitive channels in identified (1.1'-dioctadecyl-3,3,3', 3'-tetramethylindocarbocyanine methanesulfonate labeled) colon sensory neurons dissociated from adult S1 dorsal root ganglia. Channels were found in 30% (7/23) of patches in a cell-attached configuration and in 43% (48/111) of excised inside-out patches. Channels were highly selective for K(+), had a slope conductance of 54 pS in symmetrical solutions, and were blocked by tetraethylammonium, amiloride, and benzamil. Channels were also seen under Ca(2+)-free conditions. Gadolinium (Gd(3+)), a known blocker of mechanosensitive ion channels, did not block channel activity. Tetrodotoxin and 4-aminopyridine were also ineffective. The cytoskeletal disrupters colchicine and cytochalasin D reduced the percentage of patches containing mechanosensitive channels. These results indicate that rat colon sensory neurons contain K(+)-selective mechanosensitive channels that may modulate the membrane excitability induced by colonic distension.

4-Aminopyridine↗
PubMed1999

Characterization of ionic currents of cells of the subfornical organ that project to the supraoptic nuclei.

The subfornical organ (SFO) is a forebrain structure that converts peripheral blood-borne signals reflecting the hydrational state of the body to neural signals and then through efferent fibers conveys this information to several central nervous system structures. One of the forebrain areas receiving input from the SFO is the supraoptic nucleus (SON), a source of vasopressin synthesis and control of release from the posterior pituitary. Little is known of the transduction and transmission processes by which this conversion of systemic information to brain input occurs. As a step in elucidating these mechanisms, the present study characterized the ionic currents of dissociated cells of the SFO that were identified as neurons that send efferents to the SON. A retrograde tracer was injected into the SON area in eleven-day-old rats. After three days for retrograde transport of the label, the SFOs of these animals were dissociated and plated for tissue culture. The retrograde tracer was used to identify the soma of SFO cells projecting to the SON so that voltage-dependent ionic currents using whole-cell voltage clamp methods could be studied. The three types of currents in labeled SFO neurons were characterized as a 1) rapid, transient inward current that can be blocked by tetrodotoxin (TTX) characteristic of a sodium current; 2) slow-onset sustained outward current that can be blocked by tetraethylammonium (TEA) characteristic of a delayed rectifier potassium current; and 3) remaining outward current that has a rapid-onset and transient characteristic of a potassium A-type current.

Afferent Pathways↗
PubMed1999

Capsaicin sensitivity and voltage-gated sodium currents in colon sensory neurons from rat dorsal root ganglia.

DiI-labeled colon sensory neurons were acutely dissociated from S1 rat dorsal root ganglia (DRG) and studied using perforated whole cell patch-clamp techniques. Forty-six percent (54/116) of labeled sensory neurons responded to capsaicin (10(-8)- 10(-5) M) with an increase in inward current, which was a nonspecific cation conductance. Responses to capsaicin applied by puffer ejection were dependent on dose, with a half-maximal response at 4.9 x 10(-7) M; bath application was characterized by marked desensitization. Voltage-gated Na(+) currents in 23 of 30 DRG cells exhibited both TTX-sensitive and TTX-resistant components. In these cells, capsaicin induced an inward current in 11 of 17 cells tested. Of the cells containing only a TTX-sensitive component, none of six cells tested was sensitive to capsaicin. In all cells that responded to capsaicin with an increase in inward current, capsaicin abolished voltage-gated Na(+) currents (n = 21). Capsazepine (10(-6) M) significantly attenuated both the increase in inward current and the reduction in Na(+) currents. Na(+) currents were not significantly altered by adenosine, bradykinin, histamine, PGE(2), or serotonin at 10(-6) M and 10(-5) M. These findings may have important implications for understanding both the irritant and analgesic properties of capsaicin.

Action Potentials↗
PubMed1998

Mechanosensitive ion channels in putative aortic baroreceptor neurons.

Cell-attached patch-clamp experiments were performed on dissociated neurons from nodose ganglia of adult rats. Putative aortic baroreceptor neurons were identified by labeling nerve endings in the adventitia of the aortic arch with the carbocyanine dye DiI. Whereas previous experiments demonstrated the presence of mechanosensitive (MS) whole cell currents, these experiments studied single MS ion channels and examined the influence of culture conditions on their expression. Single MS channels were activated by applying negative pressure through the recording pipette. Channel openings became more frequent as the negative pressure was increased, with open probability increasing significantly above 30 mmHg. MS channels had a slope conductance of 114 pS and a reversal potential of approximately 0 mV, consistent with a nonspecific cation conductance. Channels were not affected by antagonists of voltage-gated conductances but were blocked by 20 microM gadolinium, a known blocker of MS ion channels. When nodose neurons were cocultured with aortic endothelial cells, but not aortic smooth muscle cells, the percentage of patches exhibiting MS ion channels increased significantly, suggesting that aortic endothelial cells secrete a diffusible factor that increases channel expression.

Animals↗
PubMed1998

Inhibition of calcium currents in rat colon sensory neurons by K- but not mu- or delta-opioids.

Inhibition of calcium currents in rat colon sensory neurons by kappa- but not mu- or delta-opioids. J. Neurophysiol. 80: 3112-3119, 1998. We previously reported that kappa-, but not mu- or delta-opioid receptor agonists (ORAs) have selective, potentially useful peripheral analgesic effects in visceral pain. To evaluate one potential site and mechanism by which these effects are produced, we studied opioid effects on high-voltage activated (HVA) Ca2+ currents in identified (Di-I) pelvic nerve sensory neurons from the S1 dorsal root ganglion (DRG). Results were compared with opioid effects on cutaneous neurons from L5 or L6 DRG. Di-I-labeled DRG cells were voltage clamped (perforated whole cell patch clamp), and HVA Ca2+ currents were evoked by depolarizing 240-ms test pulses to +10 mV from a holding potential of -60 mV. Neither mu-ORAs (morphine, 10(-6 )M, n = 16; [D-Ala2, N-Me-Phe4, Gly-ol5] enkephalin, 10(-6 )M, n = 12) nor delta-ORAs ([D-Pen2, D-Pen5] enkephalin, 10(-7 )M, n = 16; SNC-80, 10(-7 )M, n = 7) affected HVA Ca2+ currents in colon sensory neurons. In contrast, the kappa-ORAs U50, 488 (10(-6 )M), bremazocine (10(-6)M), and nalBzoH (10(-6 )M) significantly attenuated HVA Ca2+ currents in colon sensory neurons; effects on cutaneous sensory neurons were variable. A nonreceptor selective concentration of naloxone (10(-5 )M) and nor-BNI (10(-6 )M), a selective kappa-opioid receptor antagonist, reversed the inhibitory effect of kappa-ORAs. In the presence of N-, P-, or Q-, but not L-type Ca2+ channel antagonists, the effect of U50,488 on HVA Ca2+ currents was significantly reduced. Pretreatment with pertussis toxin (PTX) prevented the inhibition by U50,488. These results suggest that kappa-opioid receptors are coupled to multiple HVA Ca2+ channels in colon sensory neurons by a PTX-sensitive G protein pathway. We conclude that inhibition of Ca2+ channel function likely contributes in part to the peripheral analgesic action of kappa-ORAs in visceral nociception.

Animals↗
PubMed1997

Mechanical stimulation of neurites generates an inward current in putative aortic baroreceptor neurons in vitro.

We investigated the responses of putative aortic baroreceptor neurons to mechanical stimulation of their processes. Putative aortic baroreceptor neurons were identified by applying the carbocyanine dye DiI to the adventitia of the aortic arch of anesthetized rats. After at least 1 week, the nodose ganglia were removed and the neurons were cultured. Within 2-3 days, neurite outgrowth was evident on many neurons. The soma was voltage-clamped using whole cell patch clamp techniques while the neurites were deformed with pneumatic ejection of bath solution at 5-15 psi using a glass pipette (7-15 microm) positioned at least 50 microm from the neurite. Mechanical stimulation induced an inward current in 15 out of 17 putative aortic baroreceptor neurons. The magnitude of the current was related to the intensity of stimulation. The current was blocked by 20 microM gadolinium (n = 11), a reported blocker of mechanically sensitive ion channels, or by incubating the cells overnight in 10 microM phalloidin, which binds to actin filaments (n = 5). We conclude that mechanical deformation of neurites of putative baroreceptor neurons activates a mechanosensitive inward current in the soma and that the cytoskeletal actin filaments are involved in the generation of this current.

Animals↗
PubMed1997

Non-voltage-gated Ca2+ influx through mechanosensitive ion channels in aortic baroreceptor neurons.

The mechanisms underlying mechanotransduction in baroreceptor neurons (BRNs) are undefined. In this study, we specifically identified aortic baroreceptor neurons in primary neuronal cell cultures from nodose ganglia of rats. Aortic baroreceptor neurons were identified by labeling their soma with the fluorescent dye 1,1'-dioleyl-3,3,3',3'-tetramethylin-docarbocyanine (DiI) applied to the aortic arch. Using Ca2+ imaging with fura 2, we examined these BRNs for evidence of Ca2+ influx and determined its mechanosensitivity and voltage dependence. Mechanical stimuli were produced by ejecting buffer from a micropipette onto the cell surface with a pneumatic picopump, producing a shift in the center of mass of the cell that was related to intensity of stimulation. Ninety-three percent of DiI-labeled neurons responded to mechanical stimulation with an increase in [Ca2+]i. The magnitude of the increases in [Ca2+]i was directly related to the intensity of the stimulus and required the presence of external Ca2+. The trivalent cations Gd3+ or La3+ in equimolar concentrations (20 mumol/L) eliminated the K(+)-induced rises in [Ca2+]i, demonstrating that both trivalent cations are equally effective at blocking voltage-gated Ca2+ channels in these baroreceptor neurons. In contrast, the mechanically induced increases in [Ca2+]i were blocked by Gd3+ (20 mumol/L) only and not by La3+ (20 mumol/L). Stretch-activated channels (SACs) have been shown in other preparations to be blocked by Gd3+ specifically. Our data demonstrate that (1) BRNs, specifically identified as projecting to the aortic arch, have ion channels that are sensitive to mechanical stimuli; (2) mechanically induced Ca2+ influx in these cells is mediated by a Gd(3+)-sensitive ion channel and not by voltage-gated Ca2+ channels; (3) the magnitude of the Ca2+ influx is dependent on the intensity of the stimulus and the degree and duration of deformation; and (4) repeated stimuli of the same intensity result in comparable increases in [Ca2+]i. We conclude that mechanical stimulation increases Ca2+ influx into aortic BRNs independent of voltage-gated Ca2+ channels. The results suggest that Gd(3+)-sensitive SACs are the mechanoelectrical transducers in baroreceptors.

Animals↗
PubMed1995

Sodium channels in cultured neuroblastoma cells grown in high glucose or L-fucose.

Patch clamp techniques were used to record whole cell and single channel Na+ currents from NB41A3 neuroblastoma cells grown in culture. Cells were grown for two weeks in control medium or medium supplemented with 30 mM D-glucose of 30 mM L-fucose. Cells exposed to glucose or L-fucose had smaller whole cell Na+ currents than cells grown in unsupplemented medium, consistent with earlier studies (Yorek, Stefani & Wachtel, 1994). Whole cell macroscopic currents showed no change in activation or inactivation kinetics. Single channel current properties and opening probability were also unchanged. The number of [3H]saxitoxin binding sites, and therefore the total number of Na+ channels, was not reduced in cells grown in glucose or L-fucose (Yorek et al., 1994). Therefore, we conclude that some of the channels must have been rendered nonfunctional by the conditioning media. The finding that single channel properties are not altered suggests that channels become nonfunctional in an all-or-none manner.

Action Potentials↗
PubMed1995

Mechanical stimulation increases intracellular calcium concentration in nodose sensory neurons.

The cellular mechanisms involved in activation of mechanosensitive visceral sensory nerves are poorly understood. The major goal of this study was to determine the effect of mechanical stimulation on intracellular calcium concentration ([Ca2+]i) using nodose sensory neurons grown in culture. Primary cultures of nodose sensory neurons were prepared by enzymatic dispersion from nodose ganglia of 4-8 week old Sprague-Dawley rats. Whole cell [Ca2+]i was measured by a microscopic digital image analysis system in fura-2 loaded single neurons. Brief mechanical stimulation of individual nodose sensory neurons was achieved by deformation of the cell surface with a glass micropipette. In 31 of 50 neurons (62%), mechanical stimulation increased [Ca2+]i from 125 +/- 8 to 763 +/- 89 nM measured approximately 10 s after stimulation. [Ca2+]i then declined gradually, returning to near basal levels over a period of minutes. [Ca2+]i failed to increase after mechanical stimulation in the remaining 19 neurons. The mechanically-induced rise in [Ca2+]i was essentially abolished after the neurons were incubated for 5-10 min in zero Ca2+ buffer (n = 7) or after addition of gadolinium (10 microM), a blocker of stretch-activated ion channels (n = 5). The effect of gadolinium was reversed after removal of gadolinium. The results indicate that: (1) mechanical stretch increases [Ca2+]i in a subpopulation of nodose sensory neurons in culture, and (2) the stretch-induced increase in [Ca2+]i is dependent on influx of Ca2+ from extracellular fluid and is reversibly blocked by gadolinium. The findings suggest that opening of stretch-activated ion channels in response to mechanical deformation leads to an increase in Ca2+ concentration in visceral sensory neurons.(ABSTRACT TRUNCATED AT 250 WORDS)

Animals↗
PubMed1995

Mechanosensitive currents in putative aortic baroreceptor neurons in vitro.

1. Whole cell patch-clamp experiments were conducted to determine whether rat aortic baroreceptor neurons contain mechano-sensitive conductances. 2. Putative aortic baroreceptor neurons in the nodose ganglia were identified by injecting DiI onto the adventitia of the aortic arch. Nodose ganglia neurons were dissociated after > or = 1 wk. A fluorescein-conjugated tetanus toxin fragment was used to confirm that the cells labeled with DiI in culture were neurons. 3. Hypoosmotic stretch significantly increased the conductance of DiI-labeled neurons (n = 19). The reversal potential of the response was -11 +/- 1 (SE) mV. 4. In experiments on unlabeled neurons, only 7 of 13 cells showed increases in conductance. BC3H1 cells, a mouse tumor cell line, showed no changes in conductance. 5. Gadolinium (20 microM), a putative blocker of mechanosensitive channels, prevented the increase in conductance produced by hypoosmolality in seven of seven labeled cells. Equimolar concentrations of lanthanum (n = 6) and omega-conotoxin GVIA (1 microM, n = 4), which block voltage-gated calcium channels, failed to significantly affect the inward current.

Animals↗
PubMed1995

Structural versus functional modulation of the arterial baroreflex.

Structural changes in large arteries are often considered the predominant mechanism responsible for decreased baroreflex sensitivity and baroreceptor resetting in hypertension, atherosclerosis, and aging. Recent work has demonstrated that "functional" mechanisms, both at the level of the peripheral sensory endings and within the central nervous system, contribute significantly to altered baroreflex responses. We have conducted both reductive studies of mechanoelectrical transduction in cultured baroreceptor neurons and integrative studies with in vivo recordings of the activity of baroreceptor afferent fibers and efferent sympathetic nerves. Results suggest that the primary mechanism of mechanical activation of baroreceptor neurons involves opening of stretch-activated ion channels susceptible to blockade by gadolinium. Baroreceptor nerve activity is modulated by the activity of potassium channels and the sodium-potassium pump and by paracrine factors, including prostacyclin, oxygen free radicals, and factors released from aggregating platelets. Endothelial dysfunction and altered release of these paracrine factors contribute significantly to the decreased baroreceptor sensitivity in hypertension and atherosclerosis. The central mediation of the baroreflex depends on the pulse phasic pattern of afferent baroreceptor discharge. Baroreflex-mediated inhibition of sympathetic nerve activity is well maintained during pulse phasic afferent activity. Continuous, nonphasic baroreceptor discharge or a rapid (> 1.5 Hz) pulse phasic discharge results in disinhibition of sympathetic activity. This disinhibition during continuous baroreceptor input is exaggerated with aging. Thus, a defect in central mediation of the baroreflex may be a major cause of the impaired baroreflex and sympathoexcitation in the elderly. In summary, functional neural mechanisms, in addition to structural vascular changes, contribute importantly to altered baroreflex responses in normal and pathophysiological states.(ABSTRACT TRUNCATED AT 250 WORDS)

Animals↗
PubMed1995

Relative potencies of volatile anesthetics in altering the kinetics of ion channels in BC3H1 cells.

Single channel recording techniques have been used to study effects of the volatile anesthetics enflurane, halothane, isoflurane and methoxyflurane, and the gaseous agent nitrous oxide, on the properties of nicotinic channels activated by acetylcholine. Single channel currents activated by 250 nM acetylcholine were recorded from cell-attached patches of BC3H1 mouse tumor cells grown in culture. All of the potent volatile agents shortened the duration of individual opening events and caused openings to appear grouped together in bursts. The slower time constant of channel open-time distributions was decreased 50% by approximately 0.22% enflurane (0.12 mM), 0.25% isoflurane (0.10 mM), 0.30% halothane (0.16 mM), 0.076% methoxyflurane (0.21 mM) or 80% nitrous oxide (20 mM) at room temperature. Even when values were corrected to 37 degrees C, the concentrations required to decrease channel open time are less than clinical dosages. Anesthetic potency in altering channel properties was directly related to both clinical potency and lipid solubility, although those agents with greater clinical potencies required relatively higher concentrations to reduce channel open time. Results are interpreted in terms of a simple sequential channel blocking model, a sequential blocking model in which anesthetics also enhance the rate at which open channels normally close, and a cyclic blocking model in which blocked channels may close directly without having to pass back through the open state. All of the agents appeared to act in a qualitatively similar fashion and no differences were found that could account for the differing clinical profiles of the volatile anesthetics.

Anesthetics, Inhalation↗
PubMed1994

Elevated levels of glucose and L-fucose reduce 22Na+ uptake and whole cell Na+ current in cultured neuroblastoma cells.

Na+ flux was studied in cultured neuroblastoma cells grown in medium containing increased glucose or L-fucose concentrations. Chronic exposure of neuroblastoma cells to 30 mM glucose or 30 mM L-fucose caused a decrease in ouabain-sensitive and veratridine-stimulated 22Na+ uptake compared with cells cultured in unsupplemented medium. The Na+ current, determined by using whole-cell configuration of the patch clamp, was also decreased in these cells. Tetrodotoxin (3 microM), which blocked whole cell Na+ currents, also blocked veratridine-stimulated 22Na+ accumulation. Culturing cells in medium containing 30 mM fructose as an osmotic control had no effect on Na+ flux. Specific [3H]saxitoxin binding was not affected by 30 mM glucose or 30 mM L-fucose compared with cells grown in unsupplemented medium, suggesting that the number of Na+ channels was not decreased. These studies suggest that exposing cultured neuronal cells to conditions that occur in the diabetic milieu alters Na+ transport and Na(+)-channel activity.

Animals↗
PubMed1993

Physostigmine block of ion channels activated by acetylcholine in BC3H1 cells.

Single-channel recording techniques have been used to study the effects of physostigmine on the kinetics of ion channels activated by acetylcholine in BC3H1 mouse tumor cells grown in culture. Physostigmine reduced mean channel open time, with 50% reduction occurring at about 7 microM physostigmine. Although openings did not appear to occur in bursts, channel closed-time distributions exhibited a new component 7-8 msec in duration. The area of this component, but not its time constant, increased with higher concentrations of physostigmine. Results are consistent with a simple sequential channel-blocking model in which the new closed-time component represents a blocked state of the channel. Membrane hyperpolarization increased the potency of physostigmine in reducing channel open time and also prolonged the duration of the blocked state. The voltage dependence of physostigmine block was unexpected, because physostigmine is uncharged at physiological pH.

Acetylcholine↗
PubMed1992

Propofol potentiates both pre- and postsynaptic effects of vecuronium in the rat hemidiaphragm.

We have measured twitch tension in response to train-of-four stimulation in rat isolated phrenic nerve-hemidiaphragm preparations. Propofol inhibited nerve evoked twitch tension, with 50% inhibition occurring at 420 (SD 29) mumol litre-1. Although propofol 100 mumol litre-1 by itself had no effect on nerve evoked twitch tension, it potentiated the neuromuscular blocking effects of vecuronium. The decrease in train-of-four ratio with vecuronium was directly proportional to the degree of twitch inhibition, regardless of whether twitch was depressed by vecuronium alone or in combination with propofol. The finding that the train-of-four ratio was a function of the degree of block, rather than simply a function of vecuronium concentration, indicates that propofol also contributed to train-of-four fade and potentiated both pre- and postsynaptic effects of the neuromuscular blocker. The concentrations of propofol used in this study are much greater than human therapeutic blood concentrations, which are typically 25-35 mumol litre-1 (4-6 micrograms ml-1) immediately after a bolus dose of 2 mg kg-1, suggesting that neither muscle weakness nor potentiation of vecuronium-induced neuromuscular block should be of concern at propofol concentrations occurring clinically.

Animals↗
PubMed1992

Kinetics of nicotinic acetylcholine ion channels in the presence of intravenous anaesthetics and induction agents.

1. Single channel currents activated by 250 nM acetylcholine were recorded from cell-attached patches of BC3H1 mouse tumour cells grown in culture. Channels were recorded in the absence and presence of alphaxalone, diazepam, etomidate, fentanyl, ketamine, meperidine, or propofol. 2. All of the anaesthetics tested shortened channel open time but did not alter single channel current amplitude. Drug concentrations calculated to reduce the time constant of open-time distributions by 50% were 99 microM alphaxalone, 66 microM diazepam, 57 microM etomidate, 26 microM fentanyl, 15 microM ketamine, 16 microM meperidine, or 81 microM propofol. 3. Ketamine, meperidine, and propofol reduced channel open time at concentrations comparable to plasma levels attained during therapeutic use of these agents, while alphaxalone, diazepam, etomidate, and fentanyl reduced channel open time only at levels higher than those encountered clinically. 4. The potency of these drugs in decreasing channel open time appears to be directly correlated with their octanol/buffer partition coefficients. In contrast to expectations, however, agents with higher partition coefficients were less potent in altering channel open time. 5. Ketamine and meperidine produced a prominent third component in closed-time distributions, which were otherwise well described by the sum of two exponential components. Alphaxalone, diazepam, and etomidate also produced a small third component, while no additional component was seen with propofol or fentanyl. These additional components probably arise from creation of an additional closed state of the channel. 6. We conclude that these agents are not altering channel properties merely by exerting non-specific effects via the lipid bilayer and that they are probably not all acting by similar mechanisms.

Acetylcholine↗