Biomedical subjects

R A North

Publications and source records attributed to R A North.

At least 91 records · Page 5Linked to original sources

PubMed1994

Functional differences among alternatively spliced variants of Slowpoke, a Drosophila calcium-activated potassium channel.

The Slowpoke locus of Drosophila melanogaster encodes a family of alternatively spliced mRNAs which encode large conductance calcium-activated potassium channels. Variability residues in blocks of amino acids designated boxes A, C, E, G, and I. Oocytes were injected with cRNAs that had been chosen for direct functional comparison of single box differences. Single channel records from inside-out patches of oocyte membranes expressing A1 or A3 forms, E1 or E2 forms, and G2-G5 forms were analyzed and compared. The main functional difference between A1 and A3 was in unitary conductance, whereas the main difference in properties between E1 and E2 was in calcium sensitivity. Activation kinetics were different between G3 and G5, but not consistently in different A and E box backgrounds. The results indicate that alternative splicing of a common RNA precursor contributes to the functional diversity of the expressed channel. Our findings suggest that the variable region of the Slowpoke channel subunit comprises modular, yet interactive functional domains which influence the essential features of unit conductance, calcium sensitivity, and gating.

Alternative Splicing↗

PubMed1994

Cloned Ca(2+)-dependent K+ channel modulated by a functionally associated protein kinase.

Calcium-dependent potassium (KCa) channels carry ionic currents that regulate important cellular functions. Like some other ion channels, KCa channels are modulated by protein phosphorylation. The recent cloning of complementary DNAs encoding Slo KCa channels has enabled KCa channel modulation to be investigated. We report here that protein phosphorylation modulates the activity of Drosophila Slo KCa channels expressed in Xenopus oocytes. Application of ATP-gamma S to detached membrane patches increases Slo channel activity by shifting channel voltage sensitivity. This modulation is blocked by a specific inhibitor of cyclic AMP-dependent protein kinase (PKA). Mutation of a single serine residue in the channel protein also blocks modulation by ATP-gamma S, demonstrating that phosphorylation of the Slo channel protein itself modulates channel activity. The results also indicate that KCa channels in oocyte membrane patches can be modulated by an endogenous PKA-like protein kinase which remains functionally associated with the channels in the detached patch.

Adenosine Triphosphate↗

PubMed1994

Control of cationic amino acid transport and retroviral receptor functions in a membrane protein family.

A partial cDNA sequence indicated that the T lymphocyte early-activation gene (Tea) encodes a protein related to the dual-function ecotropic retrovirus receptor/cationic amino acid transporter (ecoR/CAT1), and RNA blots suggested highest Tea expression in T lymphocytes and liver (MacLeod, C.L., Finley, K., Kakuda, D. Kozad, C.A., and Wilkinson, M.F. (1990) Mol. Cell. Biol. 7, 3663-3674). The sequence of full-length Tea cDNA from liver (3683 bases) predicts a 657-amino-acid protein (CAT2 alpha) with 12-14 transmembrane domains. A long (515 base) region with six initiation codons and termination codons precedes the translation start codon. The liver Tea cDNA is identical to Tea cDNA from T lymphocytes (encoding CAT2 beta) with the exception of an apparent alternatively spliced sequence encoding a hydrophilic loop of 43 amino acids. The liver-specific sequence contains unique consensus sites for phosphorylation by cyclic AMP-dependent protein kinase and by protein kinase C. Injection of Xenopus oocytes with CAT2 alpha or CAT2 beta messenger RNA resulted in expression of Na(+)-independent cationic amino acid transport that was detected by current measurements under voltage-clamp. Although the amino acid sequences of the isoforms differ in only 21 of 43 residues with the majority of substitutions being conservative, the apparent affinity of CAT2 beta for arginine uptake was 70-fold higher than the CAT2 alpha isoform (Km 38 microM versus 2.7 mM). Neither isoform functioned as a receptor for ecotropic or amphotropic murine retroviruses. However, CAT1-CAT2 chimeric proteins that contain the first three putative extracellular loops of ecoR/CAT1 functioned as ecotropic receptors despite a diminished capacity to bind the viral envelope glycoprotein. The chimeric proteins also functioned as basic amino acid transporters with substrate affinities corresponding to the CAT2 isoform constituting the carboxyl-terminal portion. These results demonstrate that domains of these transporters can function in chimeric combinations to control viral receptor and transport functions.

Amino Acid Sequence↗

PubMed1994

Neurotensin excitation of rat ventral tegmental neurones.

1. Whole-cell patch-clamp recordings were made from ventral tegmental area neurones in rat midbrain slices in vitro. In principal cells, which are presumed to contain dopamine, neurotensin (< or = 1 microM) caused an inward current at -60 mV in thirty of forty-seven neurones and had no effect on the remainder. In secondary neurones, neurotensin caused an inward current in twelve of thirty-three cells. 2. The inward current evoked by neurotensin reached a maximum amplitude of about 80 pA, and declined over several minutes when the application was discontinued. The current was most commonly accompanied by a decrease in membrane conductance and reversed polarity at a strongly hyperpolarized potential; this reversal potential was less negative in a higher extracellular potassium concentration. Neurotensin also caused an inward current even in potassium-free internal and external solutions; this current was accompanied by a conductance increase, reversed close to 0 mV and was inhibited by reduction of the extracellular sodium concentration (from 150 to 20 mM). 3. The inward current was associated with a large increase in noise; this persisted in calcium-free solutions but was inhibited by low sodium concentration. The increase in noise was more prominent at hyperpolarized potentials. The amplitude of the unitary current underlying the increase in noise was estimated from the ratio of the variance to the mean as about 1.5 pA at -100 mV. 4. When the recording was made with an electrode containing guanosine 5'-thio-triphosphate, the steady inward current evoked by neurotensin did not reverse when the application was discontinued. When the recording electrode contained pertussis toxin, the action of neurotensin was not different although outward currents evoked by dopamine and baclofen declined with time. 5. It is concluded that neurotensin excites ventral tegmental area neurones by activating a pertussis toxin-insensitive guanosine nucleotide-binding protein. This leads to a reduction in membrane potassium conductance and an increase in membrane sodium conductance, the relative contribution of which varies from cell to cell.

Amino Acid Sequence↗

PubMed1994

Cell surface receptor for ecotropic host-range mouse retroviruses: a cationic amino acid transporter.

The cell surface receptor for ecotropic host-range murine leukemia viruses is a sodium-independent transporter for essential cationic acids. Our evidence strongly identifies this receptor as the transporter system y+, which was previously characterized by transport assays. Mutational analysis indicates that transporter activity is not necessary for viral reception. Infection of cells with ecotropic retroviruses causes only a partial down-modulation of receptor expression on cell surfaces.

Amino Acid Sequence↗

PubMed1994

Tetraethylammonium block of Slowpoke calcium-activated potassium channels expressed in Xenopus oocytes: evidence for tetrameric channel formation.

Unitary currents were recorded from inside-out membrane patches pulled from Xenopus oocytes that had been injected with RNA transcribed from a cDNA encoding the Drosophila maxi-K channel (Slowpoke). Site-directed mutagenesis was used to make cDNAs encoding channel subunits with single amino acid substitutions (Y308V and C309P). The extracellular side of the patch was exposed to tetraethylammonium (TEA) in the pipette solution; unitary currents in the presence of TEA were compared with currents in the absence of TEA to compute the inhibition. Amplitude distributions were fit by beta functions to estimate the blocking and unblocking rate constants. For wild-type channels, TEA blocked with an apparent Kd of 80 microM at 0 mV and sensed 0.18 of the membrane electric field; the voltage dependence lay entirely in the blocking rate constant. TEA blocked currents through C309P channels with a similar affinity to wild-type at 0 mV, but this was not voltage-dependent. Currents through Y308V channels were very insensitive to any block by TEA; the apparent Kd at 0 mV was 26 mM and the blockade sensed 0.18 of the electric field. Oocytes injected with a mixture of RNAs encoding wild-type and Y308V channels showed unitary currents of four discrete amplitudes in the presence of 3 mM TEA; at 40 mV these corresponded to inhibitions of approximately 80%, 55%, 25% and 10%.(ABSTRACT TRUNCATED AT 250 WORDS)

Animals↗

PubMed1994

Effect of dopamine and baclofen on N-methyl-D-aspartate-induced burst firing in rat ventral tegmental neurons.

Intracellular microelectrode recordings were made from dopamine-containing neurons of the ventral tegmental area or substantia nigra zona compacta in rat brain slices in vitro. The firing pattern of the neurons was switched from a tonic, single-spike pattern to a burst firing mode by adding N-methyl-D-aspartate (20 microM) to the superfusing solution; after adding tetrodotoxin the membrane potential underwent rhythmical oscillations of 20-40 mV at 0.5-2 Hz. Baclofen (1 microM) and dopamine (30 microM) hyperpolarized the neurons; when the potential was restored to its original level, the oscillations of potential and/or burst firing were not observed, but the tonic firing pattern was restored. These effects of baclofen and dopamine were prevented by barium (1 mM), which also prevented the membrane hyperpolarization. Oscillations of membrane current of a similar frequency were observed when the somatic membrane was voltage-clamped at -60 mV; these were also blocked by barium (1 mM). It is concluded that the oscillations in membrane potential observed with N-methyl-D-aspartate are generated predominantly at a dendritic location which is not voltage-clamped with an electrode at the soma. Baclofen and dopamine inhibit the oscillations by increasing the potassium conductance and hyperpolarizing the dendrites.

Action Potentials↗

PubMed1994

Outcome of pregnancy after the Mustard operation for transposition of the great arteries with intact ventricular septum.

OBJECTIVES: This study examined the outcome of pregnancy in patients with transposition of the great arteries and an intact ventricular septum after a Mustard operation. BACKGROUND: Before the introduction of surgical treatment, most children with transposition of the great arteries died in early infancy. A number of these patients have now reached their reproductive years. There is little information about the effect of pregnancy on cardiovascular status, particularly the ability of the right ventricle to adjust to the hemodynamic changes of pregnancy. The outcome for the offspring and their risk of congenital heart disease are also unknown. METHODS: Twenty-three female late survivors after the Mustard operation > 15 years of age were reviewed in relation to the occurrence of pregnancy and its outcome. Serial echocardiographic estimates of right ventricular volume during pregnancy were made in three local patients. RESULTS: Nine women had 15 pregnancies. They were asymptomatic before pregnancy and remained free from cardiac symptoms during each pregnancy. Right ventricular volume in the three patients studied increased during pregnancy but returned to normal at 8 to 11 weeks postpartum. There were 12 live births, 2 spontaneous abortions and 1 intrauterine death. None of the liveborn infants had evidence of congenital heart disease. CONCLUSIONS: In this small group of women with good quality late survival after a Mustard operation, pregnancy was well tolerated. We suspect that the incidence of congenital heart disease in infants of mothers with transposition of the great arteries will be at the lower end of the range for mothers with different types of congenital heart disease, but further data will be needed to confirm this.

Adult↗

PubMed1994

Uterine artery waveform as a predictor of pregnancy outcome in women with underlying renal disease.

To investigate the use of uterine artery flow velocity waveforms in predicting gestational hypertension (GH), preeclampsia (PE) and intrauterine growth retardation (IUGR), Colour Doppler ultrasound of the uterine arteries was performed at 19-24 weeks gestation in 51 women with known renal disease. On four consecutive waveforms, peak systolic (A), end-diastolic (B) and early diastolic (C) velocities were measured. Resistance index (RI) was calculated as (A-B)/A, and the severity of the waveform notch expressed as the AC ratio (A/C). Gestational hypertension was defined as a blood pressure (BP) > or = 140/90 mmHg with an increase of at least 15 mmHg in diastolic BP. PE included women with gestational hypertension and proteinuria > 300 mg/24 h or a doubling of early gestation protein excretion. IUGR was defined as a birthweight less than the 10th percentile for gestation. RI and/or AC ratio in 14 women (27%) exceeded the 90th percentile for gestational age of our low risk control population. Of the women with an abnormal test, 11 (79%) developed complications, 8 (57%) developed GH or PE, 3 (21%) IUGR alone, 2 (14%) GH and IUGR, and in one women intrauterine fetal death of an IUGR infant occurred, and 3 (21%) had an uncomplicated pregnancy. Of the women with a normal test, 34 (92%) had an uncomplicated pregnancy, and only 3 (8%) developed GH or IUGR. In summary, uterine artery waveform indices at 19-24 weeks gestation may be useful for the prediction of pregnancy complications in woman with underlying renal disease.

Adult↗

PubMed1994

Opioid actions on rat anterior cingulate cortex neurons in vitro.

Intracellular recordings were made from layer V pyramidal neurons in slices of rat anterior cingulate cortex, using electrodes that contained potassium methylsulfate and biocytin. [Met5]enkephalin (300 nM to 30 microM) reversibly reduced the amplitude of EPSPs evoked by stimulation of the subcortical white matter; the half-maximal concentration was about 800 nM. These EPSPs were blocked by (+/-)-2-amino-5-phosphonovaleric acid and 6-cyano-7-nitroquinoxaline-2,3-dione. [Met5]enkephalin also reduced the amplitude of bicuculline-sensitive IPSPs evoked by stimulation within layer V; the half-maximal concentration was about 60 nM. Both these actions of [Met5]enkephalin were mimicked by the delta-selective agonist DPDPE (Tyr-D-Pen-Gly-Phe-D-Pen) but not by the mu-selective agonist DAMGO (Tyr-D-Ala-Gly-MePhe-Gly-ol); they were blocked by the delta-selective antagonist naltrindole (apparent dissociation constant of about 0.3 nM) but not by the mu-selective antagonist CTOP (D-Phe-Cys-Tyr-D-Trp-Orn-Thr-Pen-Thr-NH2). [Met5]enkephalin did not change the amplitudes of depolarizations evoked by direct application of glutamate or hyperpolarizations evoked by direct application of muscimol (at -55 mV). Fifty percent (22 of 45) of pyramidal cells were hyperpolarized by [Met5]enkephalin; this resulted from an increase in potassium conductance, and it was mimicked by DPDPE and blocked by naltrindole. Five of seven nonpyramidal cells were hyperpolarized by [Met5]enkephalin; this was mimicked by DAMGO and blocked by CTOP.(ABSTRACT TRUNCATED AT 250 WORDS)

Animals↗

PubMed1994

Uterine artery Doppler flow velocity waveforms in the second trimester for the prediction of preeclampsia and fetal growth retardation.

OBJECTIVE: To determine the optimal method of measuring uterine artery waveforms with Doppler ultrasound when screening healthy nulliparas for subsequent development of preeclampsia and fetal growth retardation (FGR). METHODS: Color Doppler ultrasound was used to obtain uterine artery waveforms at 19-24 weeks' gestation in 458 nulliparas. In each uterine artery, the resistance index (RI), the ratio between peak systolic (A) and early diastolic (C) blood flow velocities (AC ratio) (a measure of the early notch in the uterine artery waveform), and placental position were recorded. The predictive values of these uterine artery Doppler measurements were evaluated for pregnancy complications. The major end points were preeclampsia and small for gestational age (SGA) infants. RESULTS: The best screening test for preeclampsia and SGA infants was the placental-side uterine artery RI or AC ratio above the 90th percentile for gestational age when the placenta was located on the left or right, and the highest RI or AC ratio when the placenta was midline. This method identified 51% of women with subsequent preeclampsia or SGA infants and had a positive predictive value of 29%. The test detected women with severe disease requiring delivery before 37 weeks with a sensitivity of 83% and specificity of 88%. However, the results were similar if the placental-side uterine artery RI was above an arbitrary cutoff of 0.56 or the AC ratio was above 2.05. A normal test predicted an uncomplicated pregnancy. CONCLUSIONS: Although abnormal uterine artery Doppler is associated with an increased risk of preeclampsia and FGR, the positive predictive values do not support its introduction as a routine screening test in nulliparous women.

Arteries↗

PubMed1993

Apamin increases NMDA-induced burst-firing of rat mesencephalic dopamine neurons.

Intracellular recordings made in vitro from rat midbrain dopamine neurons showed that apamin (100 nM) did not alter the regular spontaneous firing of the neurons, but it increased the occurrence of bursts of action potentials in N-methyl-D-aspartate. Apamin appeared to facilitate burst-firing induced by NMDA because, by blocking an outward calcium-activated potassium current, it increased the depolarizing action of NMDA.

Action Potentials↗

PubMed1993

Cocaine enhancement of the action of 5-hydroxytryptamine in rat cingulate cortex in vitro.

Intracellular recordings were made in vitro from pyramidal neurons in layer V of the anterior cingulate cortex of the rat. 5-Hydroxytryptamine (5-HT) reduced the amplitude of the excitatory postsynaptic potential; receptor-selective agonists and antagonists indicated that this resulted from activation of the 5-HT1B receptor. The action of 5-HT was enhanced by cocaine (300 nM-3 microM); 1 microM cocaine increased sensitivity to 5-HT about 10-fold. The results suggest that cocaine is similarly effective to potentiate the action of 5-HT in the prefrontal cortex as in other brain regions. This may contribute to its overall in vivo effects.

Animals↗

PubMed1993

Cloning and expression of a human neutral amino acid transporter with structural similarity to the glutamate transporter gene family.

A cDNA was isolated from human brain that encodes an amino acid sequence 34-39% identical to previously published glutamate transporter sequences. Injection of RNA transcribed from this cDNA into Xenopus oocytes resulted in expression of a transport activity with the properties of the neutral amino acid uptake system ASC. Superfusion of alanine, serine, and cysteine evoked sodium-dependent inward currents in voltage-clamped oocytes expressing the transporter. These currents were dose-dependent, stereospecific, and saturable, with Km values ranging from 29 to 88 microM. Northern blot analyses revealed ubiquitous expression of this gene, termed ASCT1, consistent with the general metabolic role ascribed to system ASC.

Amino Acid Sequence↗

PubMed1993

Single amino acid substitution affects desensitization of the 5-hydroxytryptamine type 3 receptor expressed in Xenopus oocytes.

5-Hydroxytryptamine type 3 receptors were expressed in Xenopus oocytes from a cloned cDNA. The peak inward current evoked by 5-hydroxytryptamine (30 microM) was linearly related to the holding potential (-100 to +20 mV) and reversed near 0 mV. The inward current (at -60 mV) declined during the continued presence of 5-hydroxytryptamine with a half-time of about 2 s; this desensitization was 20 times slower in calcium-free solution. Desensitization was markedly different in channels in which Leu286 was changed by site-directed mutagenesis; this residue is thought to lie near the middle of the M2 segment. Desensitization was faster with Phe, Tyr, or Ala in this position and slower with Thr. Phe and Thr substitutions in the equivalent position of the nicotinic acetylcholine receptor have similar effects on desensitization, suggesting that the underlying conformational change might be common to ligand-gated channels.

Amino Acid Sequence↗

PubMed1993

Opioid actions on neurons of rat lateral amygdala in vitro.

Intracellular recordings were made from neurons in the lateral nucleus of the amygdala, in a slice of rat brain that was superfused in vitro. [Met5]enkephalin (3-30 microM) and the mu receptor selective agonist DAMGO (Tyr-D-Ala-Gly-MePhe-Gly-ol; 0.3-3 microM) hyperpolarized about 50% of cells; this was blocked by naloxone and by the mu receptor antagonist CTOP (D-Phe-Cys-Tyr-D-Trp-Orn-Thr-Pen-Thr-NH2). The pA2s for naloxone and CTOP were 8.3 and 7.7, respectively. DPDPE (Tyr-D-Pen-Gly-Phe-D-Pen: delta receptor selective) and U50488 (trans-(+-)-3,4-dichloro-N-methyl-[2-(1-pyrrolidinyl)cyclohexyl] benzeneacetamide methane sulfonate; kappa receptor selective) had no effect. Synaptic potentials mediated by gamma-aminobutyric acid (GABA) acting at GABAA receptors were elicited by focal stimulation of the slice in a combination of 6-cyano-2,3-dihydroxy-7-nitroquinoxaline (10 microM) and 4-aminophosphonovaleric acid (30 microM). They were inhibited by up to 60% by DAMGO and by DPDPE. The action of DAMGO was blocked by CTOP but not by the delta-selective antagonist ICI174864 (N,N-bisallyl-Tyr-Aib-Aib-Phe-Leu-OH, Aib = aminoisobutyrate). The action of DPDPE was blocked by ICI174864 but not by CTOP. Depolarizations elicited by addition of GABA to the superfusing solution were not affected by opioids. It is concluded that activation of mu opioid receptors hyperpolarizes about 50% of lateral amygdala neurons. Activation of either mu or delta receptors also inhibits presynaptically the release of GABA.

3,4-Dichloro-N-methyl-N-(2-(1-pyrrolidinyl)-cycloh↗

PubMed1993

Membrane properties and synaptic potentials of three types of neurone in rat lateral amygdala.

1. Intracellular recordings were made from the lateral nucleus of the amygdala in tissue slices cut from rat brain and maintained in vitro. 2. Three types of neurones were distinguished according to the after-potential that followed an action potential. Type 1 cells (44%, n = 225) had depolarizing after-potentials, resulting from a calcium-dependent chloride conductance. Type 2 cells (48%) had long-lasting (> 250 ms) hyperpolarizing after-potentials and type 3 cells (8%) had shorter hyperpolarizing after-potentials. The average resting potentials of the three cell types were -78, -69 and -62 mV respectively. Intracellular labelling with biocytin showed that type 1 cells were pyramidal neurones; type 2 and type 3 cells were non-pyramidal. 3. Experiments with receptor antagonists identified synaptic potentials mediated by excitatory amino acids and by GABA (acting at GABAA receptors) in all three cell types. A longer duration inhibitory synaptic potential resulting from activation of GABAB receptors was present in type 1 (pyramidal) and type 2 cells. 4. Cholecystokinin (100 nM to 1 microM) depolarized type 2 and type 3 cells but had no effect on type 1 (pyramidal) cells. Baclofen (1-3 microM) hyperpolarized type 1 and type 2, but not type 3 cells. [Met5]enkephalin (1-10 microM) hyperpolarized only type 2 cells. 5. It is concluded that the lateral nucleus of the amygdala contains pyramidal neurones and two types of non-pyramidal neurone; these can be differentiated by membrane properties, synaptic inputs and sensitivities to transmitters.

Action Potentials↗

PubMed1993

Dopamine and baclofen inhibit the hyperpolarization-activated cation current in rat ventral tegmental neurones.

1. Whole-cell patch-clamp recordings were made from dopamine-containing ventral tegmental area neurones in slices of rat midbrain. An inward current (Ih) was activated by hyperpolarization from -60 mV. 2. Dopamine (30 microM) reduced the amplitude of Ih by 10-30% at potentials from -70 to -120 mV. The effect was concentration dependent, mimicked by the D2 agonist quinpirole, and prevented by the D2 antagonist (-)-sulpiride. Baclofen (0.3-3 microM) also inhibited Ih; this action was antagonized by 2-hydroxysaclofen but not by (-)-sulpiride. The decrease in Ih resulted from a reduction in the maximal current with no change in the voltage dependence. 3. The action of dopamine was unaffected by cadmium (200 microM), forskolin (10 microM), the adenylyl cyclase inhibitor 2',3'-dideoxyadenosine (100 microM), or by intracellular solution containing cyclic AMP (2 mM). 4. Ih was progressively reduced during the first 5-10 min of recording with electrodes containing guanosine 5'-O-(3-thiotriphosphate); after this time, dopamine had no further effect. 5. It is concluded that agonists acting at D2 receptors and GABAB receptors reduce Ih in ventral tegmental neurones.

Action Potentials↗