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Biomedical subjects

C B Gundersen

Publications and source records attributed to C B Gundersen.

At least 19 recordsLinked to original sources
PubMed1992

Expression of Ca2+ receptors in Xenopus oocytes injected with poly(A)+ mRNA from a rat calcitonin-secreting cell line.

Poly(A)+ mRNA extracted from rat calcitonin-secreting cells (rMTC 44-2) was injected into Xenopus oocytes. In mRNA-injected oocytes the intracellular Ca2+ concentration ([Ca2+]i), measured with the Ca2+ indicator dye, fura2, increased in response to an elevation of the extracellular Ca2+ ions ([Ca2+]o). In some oocytes [Ca2+]i transiently increased in high [Ca2+]o but it did not respond to the subsequent alterations of [Ca2+]o. The addition of 10 microM carbonyl cyanide m-chlorphenylhydrazone (CCCP) to the extracellular medium restored the dependence of [Ca2+]i on [Ca2+]o in such cells. It was concluded that rMTC 44-2 cells possessed a receptor which recognizes changes in [Ca2+]o and that these receptors can be functionally expressed by microinjection of messenger RNA from rMTC 44-2 cells into Xenopus oocytes.

Animals↗
PubMed1992

Suppression cloning of the cDNA for a candidate subunit of a presynaptic calcium channel.

A novel strategy, termed suppression cloning, was used to identify a 7.4 kb cDNA encoding a putative subunit of the calcium channels that regulate transmitter release at nerve endings of Torpedo californica. The 585 nt open reading frame of this cDNA encodes a polypeptide of about 21.7 kd that is essential for the expression in frog oocytes of omega-conotoxin-sensitive, dihydropyridine-resistant, calcium channels. Sequence analysis reveals that this protein is closely related to two cloned cysteine string proteins of undertermined function that were recently localized to Drosophila nerve terminals using monoclonal antibodies.

Amino Acid Sequence↗
PubMed1990

Alpha-latrotoxin triggers an increase of ionized calcium in Xenopus oocytes injected with rat brain mRNA.

When Xenopus oocytes are injected with rat brain mRNA, they acquire the ability to respond to bath applied alpha-latrotoxin. This spider venom toxin is normally highly selective for nerve endings, where its binding is associated with a high-frequency, quantal discharge of neurotransmitter. By 'transplanting' toxin acceptor sites to Xenopus oocytes, we have observed both a toxin-mediated rise in cellular ionized Ca along with the triggering of a calcium-dependent chloride channel in these cells. This approach may contribute both to a better understanding of the mechanism of action of this toxin and to efforts to clone the cDNA for this binding site.

Animals↗
PubMed1989

Direct measurement of ACh release from exposed frog nerve terminals: constraints on interpretation of non-quantal release.

1. Acetylcholine (ACh) release from enzymatically exposed frog motor nerve terminals has been measured directly with closely apposed outside-out clamped patches of Xenopus myocyte membrane, rich in ACh receptor channels. When placed close to the synaptic surface of the terminal, such a membrane patch detects both nerve-evoked patch currents (EPCs) and spontaneous quantal 'miniature' patch currents (MPCs), from a few micrometres length of the terminal, in response to ACh release from the nearest three to five active zones. 2. Chemical measurements of ACh efflux from whole preparations revealed a spontaneous release rate of 4.1 pmol (2 h)-1, and no significant difference in resting efflux between enzyme-treated and control preparations. The ratio of enzyme-treated to contralateral control muscle efflux averaged 1.17, indicating that enzyme treatment did not affect spontaneous ACh release. Vesamicol (1.7 microM), which blocks the ACh transporter in synaptic vesicles, decreased the spontaneous release of ACh to 67% of control. 3. In the absence of nerve stimulation, the frequency of single-channel openings recorded by outside-out patch probes adjacent to nerve terminals was very low (1-2 min-1), and little different at a distance of hundreds of micrometres, suggesting that if ACh was continually leaking from the terminal in a non-quantal fashion, the amount being released near active zone regions on the terminal was below the limit of detection with the patches. 4. Direct measurements of the sensitivity of the patches, coupled with calculated ACh flux rates, lead to the conclusion that the amount of ACh released non-quantally from the synaptic surface of the frog nerve terminal is less than one-tenth the amount expected if all non-quantal release is from this region of the terminal membrane. 5. Following a series of single nerve shocks or a 50 Hz train of nerve stimuli, the frequency of asynchronous single-channel openings increased for several seconds. This transient increase in channel openings was not sensitive to movement of the patch electrode a significant distance (4 microns) away from the active sites, or to manipulations previously reported to block non-quantal transmitter leakage, including addition of 10 mM-Ca2+ or 1.7 microM-vesamicol to the bath. These channel openings appear to be due to an accumulation of ACh which originated from many evoked quanta, and not the effect of locally increased non-quantal ACh release due to nerve stimulation. 6. We conclude that transmitter leakage at adult frog terminals is either localized to a source other than the synaptic surface of the nerve terminal, or released in a widespread and diffuse fashion from many sources, which may include the nerve terminal.

Acetylcholine↗
PubMed1989

Mercuric ions are potent noncompetitive antagonists of human brain kainate receptors expressed in Xenopus oocytes.

Kainate receptors are one of the major subtypes of excitatory amino acid receptors in the vertebrate central nervous system. Using Xenopus oocytes injected with RNA from human temporal cortex, it is possible to detect electrophysiologically the expression of this receptor subtype in these cells. Ions of the group IIb elements, particularly mercuric ions, are highly potent, noncompetitive inhibitors of these human brain kainate receptors. Mercury-containing sulfhydryl reagents are also very effective, irreversible blockers of the kainate-gated currents of these oocytes. The recovery of kainate-activated currents after washout of Hg2+ is slow and incomplete relative to that seen after treatment either with Cd2+ or Zn2+. Cysteine or dithiothreitol can accelerate this recovery of kainate-inducible currents after Hg2+ inhibition. Besides the toxicological implications of these results, mercury compounds may be useful for future studies of the structure and physiology of the kainate receptor-channel complex.

6-Cyano-7-nitroquinoxaline-2,3-dione↗
PubMed1988

Expression of ACh-activated channels and sodium channels by messenger RNAs from innervated and denervated muscle.

Xenopus oocytes were used to express polyadenylated messenger RNAs (mRNAs) encoding acetylcholine receptors and voltage-activated sodium channels from innervated and denervated skeletal muscles of cat and rat. Oocytes injected with mRNA from denervated muscle acquired high sensitivity to acetylcholine, whereas those injected with mRNA from innervated muscle showed virtually no response. Hence the amount of translationally active mRNA encoding acetylcholine receptors appears to be very low in normally innervated muscle, but increases greatly after denervation. Conversely, voltage-activated sodium currents induced by mRNA from innervated muscle were about three times larger than those from denervated muscle; this result suggests that innervated muscle contains more mRNA coding for sodium channels. The sodium current induced by mRNA from denervated muscle was relatively more resistant to block by tetrodotoxin. Thus a proportion of the sodium channels in denervated muscle may be encoded by mRNAs different from those encoding the normal channels.

Acetylcholine↗
PubMed1988

Barbiturates depress currents through human brain calcium channels studied in Xenopus oocytes.

Barbiturates have had wide use as sedatives, anesthetics and anticonvulsants. Among the sites implicated in the membrane action of barbiturates are the gamma-aminobutyric acidA receptor, receptors for excitatory amino acids and Ca and potassium channels. The expression in Xenopus oocytes of various ligand- and voltage-gated channels offers the opportunity for more-detailed studies of such neuroactive substances as the barbiturates. Using RNA from human temporal cortex, we obtained the expression of an omega-conotoxin-sensitive, dihydropyridine-resistant Ca channel in Xenopus oocytes. Under voltage clamp, barbiturates depressed both the peak current and the steady-state current through this Ca channel. Barbiturates had no effect on the shape of the current-voltage relation, nor did they cause a shift in the voltage-dependence of channel activation. However, both the rate of inactivation of open Ca channels, as well as the proportion of channels inactivated at steady state were increased by barbiturates. The IC50 for these effects was about 0.25 mM for the more potent barbiturates tested. These results are consistent with the hypothesis that sedative and anesthetic effects of barbiturates can be mediated in part by an action to depress Ca currents.

Animals↗
PubMed1987

Expression of an omega-conotoxin-sensitive calcium channel in Xenopus oocytes injected with mRNA from Torpedo electric lobe.

Xenopus laevis oocytes were injected with poly(A)+ RNA isolated from the electric lobe of Torpedo californica. Six to nine days after mRNA injection of the oocytes a cadmium-sensitive inward current could be detected in oocytes bathed in a calcium- and chloride-free solution containing 40 mM barium. This inward current could be distinguished from the native barium current of control oocytes by its high sensitivity to blockade by cadmium ions and its inhibition by omega-conotoxin, a peptide neurotoxin from Conus geographicus. Neither the current of control cells nor that of injected cells was detectably affected by nisoldipine (1 microM) or nitrendipine (1 microM). However, the barium current of control oocytes showed appreciably more inactivation (in the barium solution used for recording) than the omega-conotoxin-sensitive current that develops in mRNA-injected oocytes. Culturing of mRNA-injected oocytes in medium containing actinomycin D failed to prevent the appearance of the omega-conotoxin-sensitive current. These results support the conclusion that mRNA from Torpedo electric lobe is translated to produce an additional calcium channel in Xenopus oocytes. The features of this channel suggest that it may be the same type of calcium channel that controls transmitter release at nerve endings in Torpedo electroplax.

Animals↗
PubMed1987

Expression of size-selected mRNA encoding the intestinal Na/glucose cotransporter in Xenopus laevis oocytes.

The expression of the rabbit intestinal brushborder Na/glucose cotransporter has been studied in Xenopus oocytes. Poly(A)+ RNA isolated from the intestinal mucosa was injected into oocytes, and the expression of the transporter in the oocyte plasma membrane was assayed by measuring the Na-dependent phlorizin-sensitive uptake of methyl alpha-D-[14C]glucopyranoside (MeGlc). Expression of the glucose carrier was detected 3-7 days after mRNA injection, and the rate of glucose transport was proportional to the amount of mRNA injected. mRNA (50 ng) increased the maximum velocity (Vmax) of MeGlc uptake by as much as 10-fold over background. The total mRNA was fractionated by preparative agarose gel electrophoresis and each fraction was assayed for its ability to induce transport activity. The mRNA encoding the Na/glucose cotransporter was found in a single fraction of approximately 2.3 kilobases (kb), which contained 3% of the total mRNA. A similar mRNA fraction (2.0-2.6 kb) isolated from colon did not induce expression of this transporter. In vitro translation of the fractionated intestinal mRNA showed enhanced synthesis of two protein bands at 57 and 63 kDa. The mRNA encoding the cotransporter is smaller (2.3 kb) than that (2.6-2.9 kb) encoding the 55-kDa facilitated glucose carrier in human hepatoma cells and rat brain.

Animals↗
PubMed1986

Actions of pentobarbital on rat brain receptors expressed in Xenopus oocytes.

Functional receptor channels activated by GABA and other neurotransmitters were "transplanted" from rat brain to Xenopus oocytes by injecting the oocytes with total poly(A)+ mRNA isolated from rat or chick brain. Membrane currents elicited in the oocyte by GABA inverted polarity at about the chloride equilibrium potential (ca. -25 mV). Pentobarbital potentiated the GABA-activated currents, without appreciably changing the reversal potential or form of the current-voltage relationship. At low (less than 10(-5) M) concentrations of GABA, pentobarbital (100 microM) potentiated the responses by a factor of 10 or more, but responses to high (ca. 1 mM) concentrations of GABA were almost unchanged. Half-maximal activation of the response was obtained with about 3 X 10(-5) M GABA when applied alone and with about 4 X 10(-6) M GABA when applied together with 100 microM pentobarbital. At low doses of GABA, the size of the current increased as the 1.4th power of GABA concentration, but this relationship became nearly linear in the presence of pentobarbital. The potentiation of the GABA response increased linearly with concentrations of pentobarbital up to about 300 microM, reaching a maximum of about 50-fold. At higher concentrations of pentobarbital, the response to GABA declined. Relaxations of GABA-activated currents following voltage steps became slower in the presence of pentobarbital, suggesting that the open life-time of the channels was prolonged. In addition to actions on GABA-activated currents, pentobarbital itself elicited a small membrane current that inverted polarity at a potential (-10 mV) more positive than the GABA-activated current.(ABSTRACT TRUNCATED AT 250 WORDS)

Animals↗
PubMed1985

On the orientation of foreign neurotransmitter receptors in Xenopus oocytes.

Xenopus oocytes can be made to incorporate into their membrane foreign neurotransmitter receptors and voltage-activated sodium channels. In their original location the receptors are normally activated by the extracellular action of transmitter substances. Tests were made to see if some of the newly synthesized foreign receptors were inserted in the oocyte membrane with their active site facing inwards. Since intracellular injections of acetylcholine, gamma-aminobutyric acid, serotonin and kainic acid and tetrodotoxin into the oocyte failed to elicit a response, we conclude that very few, or none, of the receptor molecules expressed in the oocyte by the exogenous mRNA are inserted with the wrong orientation in the membrane.

Animals↗
PubMed1985

A transient inward current elicited by hyperpolarization during serotonin activation in Xenopus oocytes.

Activation of serotonin, glutamate or muscarinic receptors, incorporated into the membrane of Xenopus oocytes following injection of messenger RNA from rat brain, caused the development of a transient inward (Tin) current when the membrane was hyperpolarized. A detailed study was made of the Tin current induced during serotonin activation. The current is due principally to efflux of chloride ions, and is presumably activated by an influx of calcium ions, because it was blocked by removal of calcium from the bathing medium, by addition of manganese, cobalt or lanthanum, or by intracellular injection of EGTA. During application of serotonin, the amplitude of the Tin current increased slowly, and after washing it persisted for longer than the direct serotonin-induced current. The amplitude of the Tin current was sensitive to temperature and pH, and was abolished at pH 6.5 or by cooling to 12 degrees C. The Tin current may be of importance in regulating the excitability of neurons in the central nervous system.

Animals↗
PubMed1985

Intracellular Ca2+-dependent and Ca2+-independent responses of rat brain serotonin receptors transplanted to Xenopus oocytes.

Xenopus oocytes injected with messenger RNA extracted from rat brain are induced to acquire a variety of neurotransmitter receptors and voltage-operated membrane channels. Activation of the receptors to serotonin, acetylcholine (muscarinic) and glutamate elicits oscillatory membrane currents carried by chloride ions. These currents are not abolished by removing external calcium, but are completely abolished after EGTA is injected into the oocytes to chelate intracellular calcium. A smooth current response to serotonin remained in EGTA-loaded oocytes, indicating that this response does not require intracellular calcium. In contrast to the oscillatory chloride currents, the chloride currents activated by GABA or glycine are not abolished by intracellular injection of EGTA. Thus, there appear to be two classes of chloride channels one of which requires intracellular calcium to open.

Animals↗
PubMed1985

Choline acetyltransferase and acetylcholine in Xenopus oocytes injected with mRNA from the electric lobe of Torpedo.

Xenopus oocytes were injected with poly(A)+ mRNA obtained from the electric lobes of Torpedo marmorata and Torpedo ocellata, which contain the cell bodies of the neurons that innervate the electric organs. The electric lobe mRNA preparation induces the oocytes to synthesize a catalytically active form of the enzyme choline acetyltransferase (EC 2.3.1.6). Enzymatic activity is found almost exclusively in the cytoplasmic fraction of injected, but not control, oocytes. Evidence is presented that distinguishes between the induced choline acetyltransferase activity and an intrinsic carnitine acetyltransferase activity present in the oocytes. This latter enzyme is associated principally with particulate fractions of the oocyte. The level of acetylcholine, which accumulates in mRNA-injected oocytes, is relatively insensitive to pharmacological manipulations that alter the acetylcholine content of other cells. These results show that Xenopus oocytes may be used advantageously to study functional properties of polypeptides associated with presynaptic elements in the nervous system.

Acetylcholine↗
PubMed1985

Rapid diagnosis of LaCrosse encephalitis: detection of specific immunoglobulin M in cerebrospinal fluid.

An immunoglobulin M (IgM) antibody capture enzyme immunoassay (MAC-EIA) was developed for the rapid and early diagnosis of LaCrosse (LAC) virus infections. The MAC-EIA was a sensitive and specific technique for the detection of IgM antibodies to LAC virus in cerebrospinal fluid specimens and in acute-phase serum specimens. In a retrospective study, cerebrospinal fluid and acute-phase serum paired samples from 108 patients were tested by the MAC-EIA and by an IgM immunofluorescence assay. The results were compared with the original diagnosis, which was made by using a variety of classical serological tests including serum neutralization, hemagglutination inhibition, and complement fixation. Thirty patients were confirmed as having LAC virus infections; of these, 30 (100%) were diagnosed as positive by serum MAC-EIA, and 27 (90%) were positive by cerebrospinal fluid MAC-EIA. The MAC-EIA was more sensitive than the IgM immunofluorescence assay. Two patients who were not previously confirmed as positive cases were diagnosed as having LAC virus infections by the MAC-EIA. One patient who was subsequently diagnosed as having a Jamestown Canyon virus infection and two patients who were previously infected with Jamestown Canyon virus were not falsely identified as having LAC virus infections by the MAC-EIA.

Antibodies, Viral↗
PubMed1984

Glutamate and kainate receptors induced by rat brain messenger RNA in Xenopus oocytes.

Xenopus laevis oocytes injected with poly(A)+ mRNA extracted from rat brain became sensitive to serotonin, glutamate, kainate, acetylcholine and gamma-aminobutyrate. Application of these substances to mRNA-injected oocytes elicited membrane currents. The glutamate- and acetylcholine-induced currents usually showed oscillations, while the kainate current was smooth. The current oscillations during glutamate application reversed direction at about the chloride equilibrium potential (-24 mV), but the reversal potential for the kainate current was close to 0 mV. The current-voltage relation for the glutamate-induced current oscillations showed strong rectification at hyperpolarized potentials, while that for the kainate current was nearly linear. In some oocytes, glutamate elicited smooth membrane currents, with oscillations either absent, or appearing after a delay. The reversal potential of this component was close to 0 mV, and was clearly different from that of the oscillatory component. The appearance of glutamate and kainate sensitivity in the oocyte membrane is due to the translation of the foreign messenger RNA, and not to activation of the oocytes' own genome, because oocytes still become sensitive when transcription is prevented by enucleation or by treatment with actinomycin D. It appears that mRNA from rat brain contains translationally active messengers which code for various neurotransmitter receptors. When this mRNA is injected into Xenopus oocytes, the messengers are translated and receptors are inserted into the oocyte membrane, where they form functionally active receptor-channel complexes.

Animals↗
PubMed1984

Properties of human brain glycine receptors expressed in Xenopus oocytes.

Glycine and gamma-aminobutyric acid (GABA) receptors from the foetal human brain were 'transplanted' into the Xenopus oocyte membrane by injecting the oocytes with poly(A)+-mRNA extracted from the cerebral cortex. Activation of both glycine and GABA receptors induced membrane currents carried largely by chloride ions. However, unlike the GABA-activated current, the glycine current was blocked by strychnine, and was not potentiated by barbiturate. At low doses, the glycine current increased with concentration following a 2.7th power relation, suggesting that binding of three molecules of glycine may be required to open a single membrane channel. The current induced by steady application of glycine decreased with hyperpolarization beyond about -60 mV.

Animals↗