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R A Gross

Publications and source records attributed to R A Gross.

At least 37 records · Page 2Linked to original sources
PubMed1997

Dynorphin A-mediated reduction in multiple calcium currents involves a G(o) alpha-subtype G protein in rat primary afferent neurons.

We examined the effect of antisera directed at specific G-protein subtype(s) on dynorphin A (Dyn A)-mediated reduction of calcium currents in rat dorsal root ganglia (DRG) neurons. Whole cell patch-clamp recordings were performed on acutely dissociated neurons. Dyn A (1 microM)-mediated decrease in calcium currents was inhibited > 90% by the preferential kappa-receptor antagonist norbinaltorphimine. Dyn A (300-1,000 nM)-mediated reduction in calcium currents was examined during intracellular administration of antisera directed against specific regions of G(o) alpha, G(i) 1 alpha/G(1) 2 alpha, and G(i) 3 alpha subunits. Intracellular dialysis with an antiserum specific for G(o) alpha for 20 min decreased calcium current inhibition by Dyn A (1 microM) in 13 of 15 neurons by an average of 75%. Dialysis with nonimmune serum did not affect Dyn A's action to reduce calcium currents. Intracellular dialysis with either anti-G(i) 1 alpha/G(i) 2 alpha or anti-G(i) 3 alpha antisera did not affect Dyn A-induced changes in calcium currents. In the presence of the N-type calcium channel antagonist omega-conotoxin GVIA, the P-type calcium channel antagonist omega-Aga IVA, and omega-Aga MVIIC applied subsequent to the other toxins, the effect of Dyn A to reduce calcium currents was inhibited by 52, 28, and 16%, respectively. The L channel antagonist nifedipine did not affect the ability to Dyn A to inhibit calcium currents. These results suggest that in rat DRG neurons coupling of kappa-opioid receptors to multiple transient, high-threshold calcium currents involves the G(o) alpha subclass of G proteins.

Animals↗
PubMed1997

Voltage-dependent calcium channels as targets for convulsant and anticonvulsant alkyl-substituted thiobutyrolactones.

Alkyl-substituted thiobutyrolactones increase or decrease gamma-aminobutyric acidA responses at or near the picrotoxin site, but they are structurally similar to ethosuximide, which prompted us to determine the actions of thiobutyrolactones on voltage-dependent Ca++ currents. We measured Ca++ currents in cultured neonatal rat dorsal root ganglion neurons in the absence and presence of the anticonvulsant alpha-ethyl,alpha-methyl-gamma-thiobutyrolactone (alpha-EMTBL) and the convulsant beta-ethyl,beta-methyl-gamma-thiobutyrolactone (beta-EMTBL). Low-voltage-activated (T-type) currents were reduced in a concentration-dependent manner, with a maximal reduction of 26% and 30% by alpha-EMTBL and beta-EMTBL, respectively. alpha-EMTBL reduced high-voltage-activated currents in a concentration- and voltage-dependent manner: maximal responses were 7% when evoked from -80 mV, with more rapid current inactivation; 29% when evoked from -40 mV, with little effect on current inactivation. beta-EMTBL increased high-voltage-activated currents < or = 20% at 10 to 300 microM, but reduced currents at higher concentrations; the latter action was similar to that of alpha-EMTBL in its magnitude and voltage dependence. Block of N-type channels with omega-conotoxin GVIA (10 microM) reduced the effect of alpha-EMTBL and eliminated its voltage dependence. The L-type current component was also reduced by alpha-EMTBL, with little effect on P- or Q-type current components. The related compound, alpha-ethyl,alpha-methyl-gamma-butyrolactone, had no effect on Ca++ currents. We conclude that thiobutyrolactones affect voltage-dependent Ca++ currents in a concentration- and voltage-dependent manner, with greater potency on low-voltage. activated channels. Both the ring structure and the position of its alkyl substitutions determine the identity of the targeted Ca++ channel subtypes and the manner of regulation.

4-Butyrolactone↗
PubMed1995

Effects of manganese (II) on Bacillus licheniformis ATCC 9945A physiology and gamma-poly(glutamic acid) formation.

Bacillus licheniformis ATCC 9945A was cultivated in shake flasks using citrate (12 gl-1), glutamate (20 gl-1) and glycerol (80 gl-1) as carbon sources for cell growth and gamma-poly(glutamic acid) (gamma-PGA) production. The effect of the MnSO4 concentration in the medium over a range from 0.0 to 615 microns was studied. The number of viable cells increased for all concentrations of MnSO4 from approximately 10(5) to 10(9) colony-forming units (cfu) ml-1 by the early stationary phase (24 h). However, after 50 h, the cell viability decreased rapidly for relatively lower MnSO4 concentrations (0.615 and 0 microns). The utilization of carbon sources by B. licheniformis was greater for cultures containing 33.8 and 615 microns MnSO4 relative to cultures with no added MnSO4. For example, cultures with 615 microns MnSO4 utilized 37, 54 and 93% and cultures with no added MnSO4 utilized 19, 10 and 17% of glutamate, glycerol and citrate, respectively. The gamma-PGA volumetric yield increased from approximately 5 to 17 gl-1 for corresponding increases in MnSO4 concentration from 0 to 33.8 microns and then decreased at higher MnSO4 concentrations. The stereochemical content of gamma-PGA was found to vary inversely with MnSO4 concentration, and ranged from 59 to 10% L-glutamate units for MnSO4 concentrations of 0 and 615 microns, respectively. For all of the MnSO4 concentrations investigated, the gamma-PGA molecular weights decreased rapidly as the gamma-PGA volumetric yield simultaneously increased for cultivation times from 24 to approximately 50 h. Mw and Mn values after approximately 50 h cultivation times, determined by gel permeation chromatography (GPC), were 1.3 to 1.6 and 0.5 to 0.8 million g mol-1, respectively. A complex gamma-PGA molecular weight distribution that appeared bimodal by GPC analysis due to the presence of a low-molecular-weight product fraction was observed in cultures containing 33.8 and 61.5 microns MnSO4 at extended cultivation times. A high-molecular-weight fraction and the unfractionated gamma-PGA sample from the 33.8 microns MnSO4 culture contained 13 +/- 4 and 30 +/- 1% L-repeat units, respectively. A relationship between the product molecular weight and its stereochemical composition was thus established.

Bacillus↗
PubMed1994

Gamma-poly(glutamic acid) formation by Bacillus licheniformis 9945a: physiological and biochemical studies.

Cryogenically frozen vegetative cells of Bacillus licheniformis 9945a derived from young mucoid colonies were used to inoculate gamma-poly(glutamate) (gamma-PGA) production media containing L-glutamate, citrate and glycerol as carbon sources. A gel permeation chromatography (GPC) method was developed to determine gamma-PGA volumetric yield and molecular weight directly using culture filtrates. For GPC volumetric yield measurements, a calibration curve was generated using purified gamma-PGA to relate the gamma-PGA GPC peak area and polymer weight. Purified gamma-PGA was characterized by elemental analysis, 1H- and 13C-NMR spectroscopy. Cultures of B. licheniformis using all three carbon sources showed the following characteristics: cell growth mainly during the first 24 h; largest gamma-PGA volumetric productivity (approximately 0.12 gl-1 h-1) between 48 and 96 h; 11 g l-1 gamma-PGA volumetric yield by 96 h; reduction (utilization) of glycerol, glutamate and citrate during a 96 h cultivation time from 80 to 45 g l-1, 18 to 10 g l-1 and 12 to approximately 1 g l-1, respectively; a decrease in pH from 7.4 to approximately 5.5 by 42 h cultivation; acetic acid secretion into the medium at a maximum level of approximately 4.5 g l-1 and detection of the metabolite 2,3-butanediol (as acetoin) as a fermentation by-product at approximately 42 h and through a 96 h cultivation period. The presence of 2,3-butanediol indicated that the level of oxygen in the medium no longer supported a fully aerobic mode of metabolism. When the medium formulation was altered by removal of either citrate, L-glutamate or glycerol in shake flask experiments where pH was not controlled, 2.3, 9.0 and 4.0 g l-1, respectively, of gamma-PGA were formed. Variation of the medium ionic strength by the addition of up to 4% (w/v) NaCl led to the formation of gamma-PGA of relatively higher molecular weight but lower volumetric yield. Studies carried out on 5-day-old B. licheniformis cultures suggested that gamma-PGA depolymerase is intracellularly located or cell-bound. Culture filtrates showed no significant gamma-PGA depolymerase activity.

Acetates↗
PubMed1994

Genetic alteration of cyclic adenosine 3',5'-monophosphate-dependent protein kinase subunit expression affects calcium currents and beta-endorphin release in AtT-20 clonal pituitary cells.

The role of the cAMP-dependent kinase (AK) in neurotransmission was investigated by genetic alteration of AK subunit expression in AtT-20 clonal pituitary cells. We characterized and compared wild-type [AK(wt)] cells and two clones with different AK activities. The first stably expresses a gene for a mutant AK regulatory subunit (RI) that does not bind cAMP [AK(-)]; the second stably expresses a gene for the catalytic subunit (C) of AK [AK(+)]. Western blot analysis of RI and C subunit expression showed increased expression of both subunits in AK(+) and AK(-) cells relative to AK(wt), with the transfection-induced expression of one subunit producing a compensatory increase in the expression of the other. The basal AK activities varied among the cell types, with AK(+) cells possessing 3-fold higher basal AK activity than AK(wt) cells, and AK(-) cells possessing half the AK activity of AK(wt) cells. Preincubation of cultures with 300 microM 8-(4-chlorophenylthio)-cAMP increased AK activity approximately 4-fold in AK(wt) and AK(+) cells, but was without effect in AK(-) cells. Subsequent addition of 1 microM cAMP in vitro increased AK activity an additional 2- to 3-fold in all cell types. The higher basal AK activity found in AK(wt) and AK(+) cells was associated with larger whole cell calcium currents (approximately 43% and approximately 75% larger than in AK(-) cells, respectively) and faster rates of current rundown. The currents from each cell line had similar voltage-dependent and pharmacological properties, however, and [3H]PN200-110 binding was similar among the cell types. Maximal currents were evoked at clamp potentials of 0-10 mV; currents were inactivated approximately 30% in the steady state at holding potentials of -40 mV compared to -80 mV, and currents were reduced approximately 45% in the presence of nifedipine at -40 mV, but were insensitive to omega-conotoxin GVIA. AK(wt) and AK(+) cells also had higher basal and cAMP-stimulated release of beta-endorphin; control rates were approximately 50% greater, but stimulated rates were approximately 400% greater compared to those in AK(-) cells. We conclude that a greater number of calcium channels were activated by depolarization in the phosphorylated state, that current rundown was largely due to dephosphorylation, and that activation of calcium channels was coupled to the release of beta-endorphin.(ABSTRACT TRUNCATED AT 400 WORDS)

Adrenocorticotropic Hormone↗
PubMed1993

Agonists for neuropeptide Y receptor subtypes NPY-1 and NPY-2 have opposite actions on rat nodose neuron calcium currents.

1. The whole-cell variation of the patch-clamp technique was used to study the effect of neuropeptide Y (NPY) and preferential agonists for the NPY-1 and NPY-2 receptor subtypes on voltage-dependent calcium currents in acutely dissociated postnatal rat nodose ganglion neurons. 2. Both low- and high-threshold calcium current components were present. NPY altered voltage-dependent calcium currents in approximately 50% of neurons studied. NPY (0.1-100 nM, ED50 6 nM) decreased the peak amplitude of transient high-threshold calcium currents in approximately 45% of the neurons. NPY (100 nM) decreased the peak amplitude of these currents 31 +/- 5% (mean +/- SE). However, in approximately 5% of the neurons NPY (100 nM) caused a reversible and reproducible increase in transient high-threshold calcium currents of 21 +/- 4%. NPY did not affect either transient low-threshold or slowly inactivating high-threshold calcium current components. 3. Application of the C-terminal fragment NPY 13-36 (100 nM), a preferential agonist for NPY-2 receptors, reversibly decreased the peak amplitude of transient high-threshold calcium currents by 26 +/- 5% in 9 of 20 cells (45%). Application of [Pro34]-NPY (100 nM), a preferential agonist for NPY-1 receptors, reversibly increased the peak amplitude of transient high-threshold calcium currents 20 +/- 4% in 23 out of 48 neurons (48%). Six of 20 neurons (30%) responded to application of both agonists. Neither the NPY-1 nor NPY-2 agonists affected transient low-threshold or slowly inactivating high-threshold calcium current components.(ABSTRACT TRUNCATED AT 250 WORDS)

Animals↗
PubMed1992

A brief history of epilepsy and its therapy in the Western Hemisphere.

The history of epilepsy and its treatment in the western world dates back at least 4 millennia to the ancient civilization of the middle east. Past and present treatments have been empirical, usually reflecting the prevailing views of epilepsy, be they medical, theological or superstitious. Ancient physicians relied on clinical observation to distinguish between epileptic syndromes and infer their cause. Early pathophysiological theories of epilepsy correctly identified the brain as the site of the problem, but emphasized incorrect causes such as an excess of phlegm in the brain. Treatments consisted of prescribed diets or living conditions, occasional surgery such as bloodletting or skull trephination and medicinal herbs. These treatments, often ineffective, had the intellectual advantage of being based on pathophysiological principles, unlike current, more empirical, therapies. The unfortunate but widely held view of epilepsy as being due to occult or evil influences gained adherents even in the medical world during ancient times, and the later acceptance of Christianity allowed theological interpretations of seizures as well. Magical or religious treatments were more frequently prescribed as a result, practices which persist to this day. In the Renaissance an attempt was made to view epilepsy as a manifestation of physical illness rather than a moral or occult affliction, but it was during the Enlightenment that epilepsy was viewed along more modern lines, helped by advances in anatomy and pathology and the development of chemistry, pharmacy and physiology. The idea that focal irritation may cause seizures came about from clinical and experimental work, and was supported by the successful control of seizures by the (sedative) bromides and barbiturates in the late 19th century. The introduction of phenytoin showed that non-sedative drugs could be effective in controlling seizures as well, and the development of in vivo seizure models widened the scope of pharmaceutical agents tested for their efficacy against epilepsy. Increasing knowledge of the cellular mechanisms of epilepsy will, hopefully, allow the development and introduction of drugs with increasing specificity against seizure activity and the development of epilepsy.

Epilepsy↗
PubMed1992

Biodeuteration of poly(beta-hydroxybutyrate).

The formation of poly(beta-hydroxybutyrate), PHB, by Rhodobacter sphaeroides and Alcaligenes eutrophus was studied using the following carbon sources and solvents: (1), acetate in H2O; (2), D3-acetate in H2O; (3), acetate in 90 to 92% D2O; and (4), D3-acetate in 90 to 92% D2O. The growth of Rb. sphaeroides cultured under condition (2) showed no apparent deuterium isotope effect, while considerably slowed growth in the presence of D2O was observed under conditions (3) and (4). In all cases, the PHB produced under deuterium enriched conditions was of high molecular weight. Interestingly, comparatively high volumetric formation of partially deuterated PHB was obtained using culture condition (4) for A. eutrophus. Fourier transform infrared spectroscopy (FT-i.r.), pyrolysis gas chromatography mass spectrometry (PGC-m.s.), and nuclear magnetic resonance (n.m.r.) were used to establish the extent and distribution of deuterium in the PHB samples produced. Partially deuterated PHB was obtained in each case, using a deuterium enriched culture. Considerable differences in the extent and distribution of deuterium were found between micro-organisms and culture conditions.

Acetates↗
PubMed1992

The peptide CGRP increases a high-threshold Ca2+ current in rat nodose neurones via a pertussis toxin-sensitive pathway.

1. The whole-cell variation of the patch clamp technique was used to study the effect of calcitonin gene-related peptide (CGRP) on voltage-gated calcium currents in acutely dissociated rat nodose ganglion neurones and to determine if its effects were mediated via a guanine nucleotide binding (G) protein. 2. Both low- and high-threshold calcium current components were present in nodose ganglion neurones. CGRP had no effect on the isolated low-threshold current component. However, CGRP (1-1000 nM, ED50 = 50 nM) caused a concentration-dependent increase in high-threshold calcium currents. CGRP (1 microM) increased the peak of these calcium currents 21 +/- 4% over controls. 3. CGRP enhanced a transient high-threshold calcium current evoked from a holding potential of -80 mV but did not affect the slowly inactivating high-threshold current evoked from -40 mV. Multiple high-threshold calcium currents have been reported in sensory neurones. We cannot state unequivocally which high-threshold calcium current component was enhanced by CGRP. However, based on the observation that CGRP increased a transient but not the slowly inactivating high-threshold calcium current, we believe the peptide enhanced primarily the N-type calcium current component. 4. CGRP increased the maximal peak current and caused a modest negative shift of < or = 10 mV in the peak of the current-voltage (I-V) relation in three of six neurones. In the remaining three neurones the peptide increased the maximal peak current without a detectable shift in the peak of the I-V relation. 5. To determine if the CGRP-induced enhancement in calcium current was associated with an increase in calcium conductance, we studied the effect of the peptide on the instantaneous current-voltage (I-V) relation when currents were evoked at a clamp potential (Vc) of +30 mV, positive to the observed maximal current (Vc = 0 to +10 mV). CGRP increased the maximal conductance 23 +/- 4%. 6. The enhancement of calcium current by CGRP was not due to a shift in the voltage dependency of steady-state inactivation of the calcium channels. The stimulatory effect of CGRP on calcium current was evaluated by evoking currents from different holding potentials (Vh) at the same Vc (+10 mV). CGRP-induced increases in calcium currents were similar over the range of (Vh) from -60 to -110 mV, suggesting that the peptide did not alter voltage-dependent steady-state inactivation.(ABSTRACT TRUNCATED AT 400 WORDS)

Acetylcholine↗
PubMed1991

Tetrahydroaminoacridine (THA) reduces voltage-dependent calcium currents in rat sensory neurons.

Tetrahydroaminoacridine (THA) is a centrally active anticholinesterase that also interacts with neuronal K+ and Na+ channels and cardiac Ca2+ channels. The effects of THA on neuronal voltage-dependent Ca2+ channels are not known. We tested the effects of THA (25 nM-250 microM) on the Ca2+ current components of acutely dissociated rat nodose ganglion and dorsal root ganglion (DRG) neurons using the whole cell patch clamp recording technique. THA reduced the low-threshold (T) and high-threshold (N/L) Ca2+ current components in a concentration-dependent manner (IC50 approximately equal to 125 microM for T; approximately equal to 80 microM for N/L). Minimal current reduction was seen below approximately 10 microM. Our results show that THA reduces voltage-dependent Ca2+ currents in rodent sensory neurons suggesting another means by which THA may affect Ca(2+)-dependent physiologic processes.

Animals↗
PubMed1991

Comparison of effects of neuropeptide Y and norepinephrine on insulin secretion and vascular resistance in perfused rat pancreas.

Neuropeptide Y (NPY) and norepinephrine (NE) behave like cotransmitters in intrapancreatic adrenergic nerves. Therefore, in the isolated rat pancreas, we 1) studied and compared the effect of increasing concentrations of NE and NPY given alone on insulin secretion induced by 8.3 mM glucose and on pancreatic vascular flow rate and 2) investigated the effects of combinations of NPY and NE at low concentrations. NE induced a dose-dependent inhibition of insulin release between 1 and 50 nM (max 80%); beta-cells appeared sensitive to NPY at 0.1 nM, but the maximal reduction of insulin release was comparatively weak (25-30%) at 10 nM. The study of the effect of combinations of NE and NPY at different concentrations suggests that the two neurotransmitters act in an additive way to inhibit insulin secretion. NPY (0.1-10 nM) induced a marked dose-dependent reduction of pancreatic outflow rate with a biphasic pattern between 1 and 10 nM. On the other hand, at low concentrations (1 and 2 nM), NE induced a progressive increase in pancreatic outflow rate; a clear but transient decrease could only be observed at 50 nM before a secondary increase. A combined treatment with two effective concentrations of NPY (0.1 nM) and NE (1 nM) resulted in a progressive reversal by NE of NPY's vasoconstrictive effect.(ABSTRACT TRUNCATED AT 250 WORDS)

Animals↗
PubMed1990

The reduction of neuronal calcium currents by ATP-gamma-S is mediated by a G protein and occurs independently of cyclic AMP-dependent protein kinase.

We studied the effects of ATP-gamma-S on the T, N and L calcium current components of nodose ganglion neurons using the whole cell variation of the patch clamp technique. ATP-gamma-S can serve as a phosphate donor in kinase-mediated reactions, the donated phosphate group being resistant to the action of phosphatases. We therefore compared the effect of ATP-gamma-S to that of the catalytic subunit of the cyclic AMP-dependent protein kinase (AK-C), included in the recording pipette with 5 mM ATP. AK-C (50 micrograms/ml) had no effect on the T current, and caused a approximately 30% increase in currents containing the N and L components during a 20-min recording, as compared to a approximately 45% decrease in control currents. In contrast, in the presence of 2.5 mM ATP-gamma-S, T currents declined approximately 30%, and currents containing the N and L components declined to a greater extent than control currents, about 65%. In addition, the time to peak current was increased from approximately 14 ms to approximately 40 ms. This effect of ATP-gamma-S on calcium currents was similar to that of certain neurotransmitters or GTP-gamma-S, an activator of G proteins, except that the effects of ATP-gamma-S were delayed 5-7 min relative to GTP-gamma-S. The effects of both ATP-gamma-S and GTP-gamma-S were reduced or abolished in neurons treated with pertussis toxin. We conclude that AK-C regulates neuronal calcium currents, presumably by phosphorylation of channels or associated proteins, and that the ATP-gamma-S-induced reduction of calcium currents cannot be due to its serving as a phosphate donor for endogenous AK.(ABSTRACT TRUNCATED AT 250 WORDS)

Adenosine Triphosphate↗
PubMed1990

Valproic acid selectively reduces the low-threshold (T) calcium current in rat nodose neurons.

Valproic acid (VPA) is an antiepileptic drug used in the treatment of a wide variety of human seizures including generalized absence (GA) (petit mal) seizures. The mechanism of action of VPA in controlling GA seizures is not known. We tested the effects of VPA on the Ca2+ current components of acutely dissociated rat nodose ganglion neurons. VPA reduced the low-threshold (T) Ca2+ current at clinically relevant concentrations but had no effect on the high-threshold (N and L) current components. The effect on T current was concentration-dependent and most apparent at peak current. There was little effect seen on late current. VPA did not affect the rate or voltage-dependency of T current activation. The selective reduction of T current may be a means by which VPA is effective in controlling GA seizures.

Animals↗
PubMed1990

Plastics from bacteria and for bacteria: poly(beta-hydroxyalkanoates) as natural, biocompatible, and biodegradable polyesters.

Hence, PHB belongs to the family of poly(beta-hydroxyalkanoates), PHA, all of which are usually formed as intracellular inclusions under unbalanced growth conditions. Recently, it became of industrial interest to evaluate PHA polyesters as natural, biodegradable, and biocompatible plastics for a wide range of possible applications such as surgical sutures or packaging containers. For industrial applications, the controlled incorporation of repeating units with different chain lengths into a series of copolymers is desirable in order to produce polyesters with a range of material properties because physical and chemical characteristics depend strongly on the polymer composition. Such "tailormade" copolymers can be produced under controlled growth conditions, in that if a defined mixture of substrates for a certain type of microorganisms is supplied, a well defined and reproducible copolymer is formed.

Bacteria↗
PubMed1990

Dynorphin A and cAMP-dependent protein kinase independently regulate neuronal calcium currents.

The kappa-selective opioid peptide dynorphin A (DYN) inhibits neuronal adenylate cyclase activity and reduces neuronal voltage-dependent calcium currents. It is not yet known, however, whether the regulation of calcium channel activity is dependent on or independent of the adenylate cyclase/cAMP system. We used the whole-cell variation of the patch clamp technique to show that DYN reversibly reduced, in a naloxone-sensitive manner, calcium currents in acutely dissociated rat nodose ganglion neurons. DYN slowed the rate of current activation and had a greater effect on currents evoked from relatively negative holding potentials. These actions were mimicked by guanosine 5'-[gamma-thio]triphosphate, which activates GTP-binding proteins (G proteins), and were blocked by pretreatment with pertussis toxin, which inactivates Gi- and Go-type G proteins. In contrast, calcium currents recorded in the presence of the catalytic subunit of the cAMP-dependent protein kinase (AK-C), included in the recording pipette, increased in magnitude throughout the recording. DYN was applied to neurons before and after the effect of AK-C became apparent; the reduction of calcium currents by DYN was greater in the presence of AK-C than in its absence. We conclude that the acute reduction of neuronal calcium currents by DYN occurred by means of activation of pertussis toxin-sensitive Gi- or Go-type G proteins. The persistence of the action of DYN in the presence of AK-C indicates, however, that this effect was independent of a reduction of the activity of the adenylate cyclase/cAMP system and suggests in addition that phosphorylated channels may be preferentially inhibited by DYN.

Adenylate Cyclase Toxin↗
PubMed1990

The cyclic AMP-dependent protein kinase catalytic subunit selectively enhances calcium currents in rat nodose neurones.

1. The whole-cell variation of the patch clamp technique was used to study the effect of the purified catalytic subunit of the cyclic AMP-dependent protein kinase (A kinase catalytic subunit: AK-C) on the calcium current components of acutely dissociated rat nodose ganglion neurones. 2. The transient low-threshold calcium current component (T) was stable during whole-cell recording. In contrast, currents containing the transient high-threshold (N) and slowly inactivating high-threshold (L) current components declined steadily after stabilization of the currents during the first 5-7 min of recording. When AK-C was included in the recording pipette at physiological concentrations (50 micrograms/ml, approximately 1 microM), currents containing the N- and L-components increased in magnitude beginning 7-9 min after patch rupture, but there was no effect on the isolated T-current. The current-voltage relation of the T-current component was similar to controls, but the current-voltage relation for the N- and L-current components was shifted slightly to more depolarized clamp potentials (Vc), approximately 10 mV. 3. The effect of AK-C on currents containing the N- and L-currents was concentration dependent. There was no effect of 0.1 microgram/ml AK-C, the lowest concentration tested. Currents evoked from holding potentials (Vh) = -80 mV increased 5-10% during a 20 min recording in the presence of 1 microgram/ml AK-C and 30-35% in the presence of 50 micrograms/ml AK-C. In contrast, currents evoked from Vh = -40 mV increased 5-10% in the presence of either 1 or 50 micrograms/ml AK-C. The increase in current magnitude was associated with an increased rate of current inactivation and was evident particularly in currents evoked from Vh = -80 mV. 4. These effects were blocked by prior incubation of AK-C (1 microgram/ml) with a specific peptide inhibitor (protein kinase inhibitor peptide, PKIP; 0.2 mg/ml). 5. We evoked calcium currents using very long (1 s) voltage commands and modelled the traces using a multiexponential function in order to determine the effects of AK-C on the N- and L-current components. The (curve-fitted) N- and L-current components each declined approximately 50% during a 20 min recording in control neurones.(ABSTRACT TRUNCATED AT 250 WORDS)

Animals↗
PubMed1990

Neuropeptide Y reduces calcium current and inhibits acetylcholine release in nodose neurons via a pertussis toxin-sensitive mechanism.

1. The effect of neuropeptide Y (NPY) on voltage-dependent calcium currents was studied in acutely dissociated rat vagal afferent (nodose) neurons by the use of both intracellular single-electrode and whole-cell patch-clamp techniques. 2. Nodose neurons exhibited three calcium current components similar to the transient low-threshold (T), slowly inactivating high-threshold (L), and the transient high-threshold (N) currents previously described in dorsal root ganglion neurons (Nowycky et al. 1985). The characteristics of calcium current components were similar for the two recording techniques except that the inactivation time constants (tau i) were two- to threefold larger at 22 degrees C (whole-cell patch clamp) than at 35 degrees C (single-electrode voltage clamp). 3. NPY (0.1-100 nM, ED50 4 nM) produced a concentration-dependent reduction in calcium currents with the use of both recording techniques. NPY (100 nM) had no effect on T and L currents but reduced the combined N/L current 31 +/- 6% in 47% of the cells tested. Current traces were also analyzed by multiexponential curve fitting to determine amplitudes and inactivation time constants (tau i). NPY selectively reduced the amplitude of the curve-fitted N current component 45 +/- 8% but had no effect on any of the tau i. The effect of NPY to reduce calcium current was blocked in the presence of gadolinium (1 microM), a putative N channel antagonist. Pretreatment of cultures with pertussis toxin (PTX) (100 ng/ml) for 16-24 h blocked the effect of NPY. 4. NPY reduced the peak current without changing the voltage dependence of the peak current-voltage relation.(ABSTRACT TRUNCATED AT 250 WORDS)

Acetylcholine↗