Biomedical subjects

R B Clark

Publications and source records attributed to R B Clark.

At least 73 records · Page 4Linked to original sources

PubMed1994

Differential expression of the beta-adrenergic receptor modifies agonist stimulation of adenylyl cyclase: a quantitative evaluation.

The effect of varying levels of beta 2-adrenergic receptor (beta AR) expression on the capacity of the receptor to activate adenylyl cyclase through regulatory G proteins has been systematically explored in this paper, using differential expression of hamster and human beta AR in L cells. Equations have been developed based on the cycle of G protein activation first proposed by Cassel and Selinger and the mobile receptor model, which assumes that hormone-bound beta AR can stimulate a number of different adenylyl cyclase moieties through the G protein during a single cycle of activation. These equations predict the relationship of receptor number to the EC50 (potency) and Vmax (efficacy) for adenylyl cyclase activation. L cell clones were selected with stable expression of the beta AR over a 2000-fold range of levels (from 5 to 10,000 fmol/mg of membrane protein). Experimentally determined values for the EC50 and the Vmax for epinephrine stimulation of adenylyl cyclase over the entire range of receptor levels were found to be in excellent agreement with predictions of the traditionally accepted models. A method is introduced that allows calculation of beta AR coupling efficiency while taking into account the effect of variable receptor levels. The approach provides a quantitative means for the determination of coupling efficiency of the receptor/G protein/adenylyl cyclase system over wide variations in receptor levels and allows for a rational comparison of coupling efficiencies of wild-type and mutant receptors when receptor levels differ.

Adenylyl Cyclases↗

PubMed1993

Okadaic acid induces both augmentation and inhibition of beta 2-adrenergic stimulation of cAMP accumulation in S49 lymphoma cells.

To address the role of protein phosphatases in regulating hormonal responses in mammalian cells, we investigated the effects of okadaic acid, a potent inhibitor of protein phosphatases 1 and 2A, on epinephrine and prostaglandin E1 stimulation of cAMP accumulation and adenylyl cyclase in S49 WT and kin- lymphoma cells. Depending on the dose and time of okadaic acid pretreatment of both cell lines, there were two distinguishable effects on cAMP accumulation, an augmentation and an inhibition. The augmentation occurred rapidly (t1/2 < 1 min), was maximal with 3 microM okadaic acid, and was observed with concentrations of okadaic acid as low as 0.3 microM. Prolonged (t1/2 of 5-15 min) pretreatment of cells with okadaic acid caused an inhibition of epinephrine-stimulated cAMP accumulation, which was characterized by a 2-3-fold increase in the EC50 for the response to epinephrine. The EC50 for the okadaic acid-mediated inhibition was similar to that for the augmentation. In assays of adenylyl cyclase in membrane fractions prepared from okadaic acid-pretreated cells the inhibitory, but not the stimulatory, effects of okadaic acid pretreatment were observed. The data demonstrate that protein phosphatases play an important role in regulating adenylyl cyclase and suggest that cAMP-dependent protein kinase is not involved in either of its actions.

Adenylyl Cyclases↗

PubMed1993

Comparison of susceptibility test methods to detect penicillin-resistant Streptococcus pneumoniae.

The detection of penicillin-resistant Streptococcus pneumoniae was assessed by six different methods: agar dilution, oxacillin screen by disk diffusion, E-test, and three overnight microdilution test methods that included commercial panels from MicroScan and Micro Media and in-house-made conventional panels using a commercial Haemophilus test medium (HTM) broth. Of the 52 pneumococcal isolates tested, 12 were resistant, 16 were relatively resistant, and 24 were susceptible to penicillin as defined by the reference agar dilution method. The oxacillin screen detected as resistant all 28 resistant and relatively resistant strains. The percentage of penicillin-resistant isolates detected by each minimum inhibitory concentration (MIC) test method was as follows: E-test (100%), Micro Media (75%), MicroScan (0%), and HTM (0%). With the relatively resistant isolates, the detection percentage was as follows: E-test (88%), Micro Media (94%), MicroScan (69%), and HTM (69%). In conclusion, the E-test and Micro Media MIC tests are acceptable confirmatory tests for detecting penicillin resistance among S. pneumoniae isolates.

Culture Media↗

PubMed1993

Heterogeneity of action potential waveforms and potassium currents in rat ventricle.

OBJECTIVE: The ionic mechanisms for differences in action potential waveforms in rat left ventricle were studied by recording L-type Ca2+ current, transient outward K+ current, and inwardly rectifying background K+ current in single myocytes. METHODS: Single cells were obtained from adult rat hearts by enzymatic dispersion of tissue segments from the epicardium at the apex and the endocardium at the base of the left ventricle. Whole cell voltage clamp methods together with cell shortening measurements were used to identify the K+ currents involved in early and late repolarisation and to correlate changes in action potential shape with inotropic responses. 4-Aminopyridine was used to block the transient outward K+ current, I(t), to evaluate the contribution of this current to repolarisation. RESULTS: Action potential recordings demonstrated that cells from endocardial tissue at the base of the left ventricle have a considerably longer action potential than those from epicardial tissue at the apex. 4-Aminopyridine had a much more pronounced action potential lengthening and inotropic effects on cells from epicardium than on myocytes from endocardium suggesting that I(t) is larger in the epicardium. Voltage clamp measurements confirmed this. In contrast, the L-type Ca2+ current, the resting membrane potential, and the inwardly rectifying background K+ current were very similar in these two regions of left ventricle. CONCLUSIONS: One significant factor contributing to the heterogeneity of action potential waveforms in rat left ventricle is a differential distribution of a Ca+ independent transient outward K+ current, I(t). Regional differences in action potential duration have important implications for the gradient of repolarisation in rat left ventricle, for the genesis of the T wave of the electrocardiogram, and for both electrical and mechanical restitution (refractoriness).

4-Aminopyridine↗

PubMed1993

Activity of meropenem and other antimicrobial agents against uncommon gram-negative organisms.

The in-vitro activity of meropenem and other antimicrobial agents was tested by agar dilution against 113 uncommon Gram-negative pathogens. Both meropenem and ceftriaxone showed the most activity against the test isolates. The potency of meropenem was greater than imipenem against Pasteurella multocida, Eikenella corrodens, Haemophilus parainfluenzae, Moraxella spp., Kingella spp., and Actinobacillus actinomycetemcomitans. Further work is necessary to assess the in-vivo activity of meropenem against these Gram-negative isolates.

Anti-Bacterial Agents↗

PubMed1993

Regulation of unloaded cell shortening by sarcolemmal sodium-calcium exchange in isolated rat ventricular myocytes.

1. Regulation of unloaded cell shortening and relaxation by sarcolemmal Na(+)-Ca2+ exchange was investigated in rat ventricular myocytes. Contraction of single cells at 22 +/- 1 degrees C was measured simultaneously with membrane current and voltage using the whole-cell voltage clamp technique in combination with a video edge-detection device. 2. The extent of mechanical activation (cell shortening amplitude) was strongly dependent on diastolic membrane potential over the voltage range -140 to -50 mV. This voltage sensitivity of contraction was abolished completely when a recently described inhibitory peptide of the cardiac Na(+)-Ca2+ exchanger (XIP, 2 x 10(-5) M) was present in the recording pipette, demonstrating that in rat ventricular cells Na(+)-Ca2+ exchange is modulated by diastolic membrane potential. 3. Possible influences of Na(+)-Ca2+ exchange on contraction were studied from a holding potential of -80 mV. Depolarizations (-50 to +60 mV) resulted in a bell-shaped shortening-voltage (S-V) relationship. These contractions were suppressed completely by either Cd2+ (10(-4) M) or verapamil (10(-5) M), but remained unchanged during superfusion with tetrodotoxin (TTX, 1.5 x 10(-5) M), when [NA+]o was reduced from 140 to 10 mM by substitution with either Li+ or Cs+ ions or when pipette Na+ was varied between 8 and 13 mM. XIP (2 x 10(-5) M) increased the magnitude and duration of twitch contractions, but had no effect on the shape of the S-V relationship. Thus, the Ca2+ current but not the Na+ current or Ca2+ influx due to reversed Na(+)-Ca2+ exchange can release Ca2+ from the sarcoplasmic reticulum (SR) under these experimental conditions. 4. The effect of the rate of repolarization on cell shortening was studied under voltage clamp by applying ramp waveforms immediately following the depolarizations which activated contraction. Although slowing of the rate of repolarization had no effect on the first contraction following a train of conditioning depolarizations, a positive inotropic effect developed thereafter. 5. Caffeine (10 mM) was applied to determine whether Na(+)-Ca2+ exchange and/or Ca2+ sequestration/buffering by the sarcoplasmic reticulum were primarily responsible for these inotropic effects. In the presence of caffeine the positive inotropic effect developed fully during the first test depolarization. Changes in the rate of repolarization had much less effect on shortening in cells dialysed intracellularly with XIP (2 x 10(-5) M). In combination, these results suggest that the changes in the inotropic effects resulting from changes in rate of repolarization may be due to altered loading and release of Ca2+ from the SR.(ABSTRACT TRUNCATED AT 400 WORDS)

Animals↗

PubMed1993

Role of sodium-calcium exchange in activation of contraction in rat ventricle.

1. The functional role of reverse Na(+)-Ca2+ exchange in the activation of contraction of rat ventricular myocytes has been studied. Mechanical activity of single cells, measured as unloaded cell shortening, was recorded simultaneously with membrane current and voltage using a single microelectrode voltage clamp and a video edge detection device. 2. The voltage dependence of contraction was studied by applying trains of depolarizations. At test potentials between +20 and +80 mV (under conditions where large outward currents were activated) a plateau on the shortening vs. voltage (S-V) relationship was observed. Significant cell shortening also occurred at test potentials between -70 and -40 mV; and these contractions were accompanied by large inward Na+ currents. We have investigated the ionic mechanisms for three components of the S-V relation in rat ventricle: (i) shortening which occurs between -70 and -40 mV and is thought to be dependent on the sodium current; (ii) phasic contractions in the voltage range -40 to +40 mV where the L-type Ca2+ current is present; (iii) the plateau of the S-V relation at strongly depolarized voltages where reverse Na(+)-Ca2+ exchange may occur. 3. Experiments in which two independent microelectrode impalements were made in a single myocyte showed that during activation of contraction at test potentials between -70 and -40 mV, and during very large depolarizations (+20 to +80 mV), there were significant deviations of the measured membrane potential from the applied voltages. Activation of cell shortening in these voltage ranges could be eliminated by electronic series resistance compensation, which significantly reduced these voltage errors. Consistent with these findings, when tetrodotoxin (TTX) and 4-aminopyridine (4-AP) were used to block inward Na+ and transient outward K+ currents, respectively, no significant voltage errors were present and a bell-shaped shortening-voltage (S-V) relationship was obtained. 4. When Na+ and K+ currents were blocked, depolarizations from holding potentials of either -80 or -50 mV demonstrated that the threshold for activation of contraction was about -30 mV, and that the voltage dependence of peak shortening was very similar to that of the L-type Ca2+ current (ICa,L). These contractions were suppressed completely by either Cd2+ or ryanodine, showing that activation of cell shortening was due to Ca2+ influx through L-type channels which induced release of Ca2+ from the sarcoplasmic reticulum (SR). No T-type calcium currents were observed.(ABSTRACT TRUNCATED AT 400 WORDS)

4-Aminopyridine↗

PubMed1993

Identification of nonselective cation channels in cultured adult rat alveolar type II cells.

There is evidence supporting the role of active transport of Na+ in the resolution of pulmonary edema, but the exact cellular mechanism(s) underlying this process remain unknown. This study demonstrated the presence of ion channels on adult rat alveolar type II cells that might be associated with this active transport of Na+. Patch-clamp techniques were used to characterize a nonselective cation channel in adult rat alveolar type II epithelial cells held in culture for 24 to 72 h. Single-channel currents were recorded from inside-out, cell-free membrane patches. The most common type of single channel had a linear slope conductance of 20.4 +/- 0.6 pS (n = 22) in symmetrical NaCl (150 mM) solutions. The channel was approximately equally permeable to Na+ and K+ ions (PK/PNa = 1.15) and was highly selective for cations (PCl/PNa < 0.05). Channel activity was Ca(2+)-dependent, and it required at least 10 microM Ca2+ on the cytosolic side of an inside-out patch to activate the channel. Amiloride (1 to 10 microM), a Na+ channel blocker in epithelial tissue, reduced the steady-state open probability of the channel 10-fold but had no significant effect on the magnitude of the single-channel conductance. Single channels with similar properties were not found in cultured rat alveolar macrophages. The possible role of this amiloride-sensitive, nonselective cation channel in Na+ transport and lung liquid clearance is discussed.

Amiloride↗

PubMed1993

Beta 2-adrenergic receptor mutants reveal structural requirements for the desensitization observed with long-term epinephrine treatment.

In the present study wild-type and various mutant hamster beta 2-adrenergic receptors (beta ARs) expressed in L cells were used to examine potential molecular mechanisms involved in the desensitization of hormonal stimulation of adenylyl cyclase observed after long term exposure to low concentrations of epinephrine. The mutant beta ARs included deletion mutants, D(259-262)beta AR and D(343-348)beta AR, that lack the consensus sites for cAMP-dependent protein kinase (cAPK) and protein kinase C (PKC) and a truncation mutant, T(354)beta AR, that lacks the putative consensus sites for beta AR kinase. Epinephrine stimulation of adenylyl cyclase was desensitized in all four cell lines after growth for 24 hr in the presence of 3 nM epinephrine, and this desensitization was characterized by a 3-4-fold increase in the EC50 for epinephrine stimulation of adenylyl cyclase. In addition, the Vmax was significantly decreased in the cells with the wild-type beta AR and the D(343-348)beta AR. The desensitization was not masked by high concentrations of magnesium and was accompanied by a 40-70% down-regulation of beta ARs. In the cells treated with 3 nM epinephrine, prostaglandin E1 stimulation of adenylyl cyclase was decreased 11% in cells with the wild-type beta AR and forskolin stimulation was decreased 25-36% with all but the D(259-262)beta AR mutant. These results demonstrated that phosphorylations of the cAPK/PKC consensus sites and the serine- and threonine-rich segment of the carboxyl-terminal tail of the beta AR were not required for the desensitization caused by 3 nM epinephrine, thus further differentiating it from cAPK- or PKC-mediated desensitization and the desensitization attributed to beta AR kinase.

Adenylyl Cyclases↗

PubMed1992

Comparison of CNS homing pattern among murine TH cell lines responsive to myelin basic protein.

A myelin basic protein (MBP)-reactive TH cell line capable of inducing experimental allergic encephalomyelitis (EAE), and a MBP-reactive TH cell clone that does not cause EAE were labeled with a fluorescent vital dye, and transferred into naive syngeneic SJL/J mice. Animals were killed before the appearance of symptoms (3 and 4 days post-injection). Sections obtained from the spleen, spinal cord and brain of both groups of animals were examined by fluorescence microscopy to localize labeled TH cells. At all time points examined, the spleens of both groups contained innumerable labeled cells. The spinal cords and brains of animals that had received EAE-causing cells had a basal level of 20 labeled cells/cm2 at 3 days; this number increased rapidly to 150 cells/cm2 in the spinal cord at 4 days. Perivascular infiltrates and small foci of astrogliosis were already apparent in this group 3 days after injection. The spinal cords and brains of animals that had received the non-EAE-causing TH cells contained 50 labeled cells/cm2 at 3 days. The density of these transferred cells, as compared to that of the EAE-causing cells, suggested that they have an unaltered CNS-homing capability. However, by 4 days, the number of non-EAE-causing labeled cells had returned to near basal level. Our findings suggest that discrimination between disease and non-disease causing MBP-responsive TH cells occurs within the first 3 days following transfer, requires the presence in the CNS of a limited number of TH cells, and depends on yet unidentified TH cell factor(s).

Animals↗

PubMed1992

Antibiotic susceptibilities of the Vibrionaceae to meropenem and other antimicrobial agents.

The in vitro activity of meropenem was compared with imipenem and other selected antimicrobial agents against 115 isolates from the family Vibrionaceae. No resistance was observed with meropenem or imipenem against these isolates. However, meropenem was generally four- to 16-fold more active than imipenem against the aeromonads and Vibrio cholerae. Meropenem showed excellent in vitro activity against the Vibrionaceae and may be useful for eradicating infections produced by these organisms.

Aeromonas↗

PubMed1992

From Fanny to Fernand: the development of consumerism in pain control during the birth process.

Obstetric anesthesia has been the object of public interest and patient advocacy from its introduction in the 1840s to the present. Early arguments concerned the significance of pain in childbearing and became a popular issue involving physicians, clergymen, and journalists. The first obstetric anesthesia, either, given in the United States was administered in Cambridge, Massachusetts, in 1847. For the next several decades general anesthesia was the only feasible means of relieving labor pain. At the turn of the century a combination of scopolamine and morphine was introduced in the United States by a popular women's magazine; the National Twilight Sleep Association was launched. After the decline of this movement, the "natural" childbirth method came into national prominence. In spite of their differing pharmacologic characteristics, there are sociologic parallels between the patient advocacy of Twilight Sleep and that of psychoprophylaxis. This study focuses on the public perception of these movements, which were begun by physicians and subsequently endorsed by militant lay groups.

Anesthesia, Obstetrical↗

PubMed1992

Multicentre study on antibiotic susceptibilities of anaerobic bacteria to cefoperazone-sulbactam and other antimicrobial agents.

The antibiotic susceptibilities of 241 anaerobic bacteria recovered from six geographic sites in North America were tested by agar dilution to cefoperazone-sulbactam and other drugs. Of the 189 Bacteroides fragilis group isolates, only one was resistant to cefoperazone-sulbactam (0.5%) or ampicillin-sulbactam (0.5%), and none was resistant to ticarcillin-clavulanate or chloramphenicol. No resistance to cefoperazone-sulbactam was observed among the other Bacteroides spp., Clostridium spp., or Peptostreptococcus spp. Resistance to cefoperazone-sulbactam is not commonly observed against anaerobic bacteria recovered from different geographical sites across North America.

Anti-Bacterial Agents↗

PubMed1992

Role of an inwardly rectifying potassium current in rabbit ventricular action potential.

1. Whole-cell voltage-clamp measurements were made of the time- and voltage-dependent properties of the inwardly rectifying background potassium current IK1, in single myocytes from rabbit ventricle. The main goal of these experiments was to define the role of IK1 in the plateau and repolarization phases of the action potential (AP). 2. Action potentials from single ventricular myocytes were used as the command signals for voltage-clamp measurements. In these 'action potential voltage-clamp' experiments, IK1 was isolated from other membrane currents by taking the difference between control currents and currents in K(+)-free bathing solution. The results show that IK1 is small during the plateau, but then rapidly increases during repolarization and declines in early diastole. 3. Evidence of an important functional role for IK1 in AP repolarization was obtained by comparing the magnitude of IK1 and the rate of change of membrane potential (dVm/dt) in the same cell during the AP. The time courses of IK1 and dVm/dt during the AP were closely correlated, indicating that IK1 was the principal current responsible for final repolarization. 4. Rectangular voltage-clamp steps were used to study time- and voltage-dependent changes in IK1 at membrane potentials corresponding to the repolarization phase of the AP. 'Slow' relaxations or tail currents, lasting 100-300 ms, were consistently recorded when the cell was repolarized to potentials in the range -30 to -70 mV, following depolarizations between +10 and -10 mV. 5. The close correlation between the magnitude of the steady-state IK1 (in an external K+ concentration of 5.4 mM), which was outward for membrane potentials in the range -30 to -70 mV, and the magnitude of the tail currents, suggests that they resulted from a slow increase, or reactivation, of IK1. 6. The component of the slow tails due to reactivation of IK1 can be separated from a previously described component due to Na(+)-Ca2+ exchange since the IK1 component: (i) does not depend on the presence of the calcium current, ICa; (ii) can be recorded when internal EGTA (5 mM) suppresses large changes in [Ca2+]i; (iii) does not depend on the Na+ electrochemical gradient; (iv) is abolished in K(+)-free external solution; and (v) is not present in rabbit atrial myocytes, in which IK1 is very small. 7. The time- and voltage-dependent properties of IK1 revealed by these tail current experiments suggest that the measured magnitude of IK1 will be dependent on the voltage-clamp protocol.(ABSTRACT TRUNCATED AT 400 WORDS)

Action Potentials↗

PubMed1992

Hyperthyroidism selectively modified a transient potassium current in rabbit ventricular and atrial myocytes.

1. Transient outward potassium currents (I(t)) were compared in single cardiac myocytes obtained from normal and hyperthyroid rabbits. Currents were recorded using the suction electrode whole-cell voltage clamp technique. 2. In ventricular myocytes from hyperthyroid animals (at 22 degrees C and a stimulation rate of 0.2 Hz), I(t) was 4- to 5-fold larger than in normal myocytes, in a potential range of -20 to +60 mV. As in normal myocytes, I(t) in hyperthyroid myocytes was calcium insensitive, and was more than 90% suppressed by 2 mM 4-aminopyridine. 3. The increase in I(t) was observed over a wide range of stimulation rates, even at rates sufficiently slow to enable complete reactivation of the I(t) channels. However, there was a major change in the rate dependence of I(t) in hyperthyroid myocytes, with significant I(t) current still present at rates (e.g. 1-2 Hz) at which it is normally completely suppressed. 4. The augmentation of I(t) in the hyperthyroid myocytes could not be accounted for by changes in the voltage dependence or the kinetics of channel activation or inactivation. There was no change in the reversal potential of I(t), implying no change in the selectivity of the channel. 5. Single-channel activity was recorded using the cell-attached mode of recording. In myocytes from hyperthyroid rabbit we observed the following: (a) active patches (often containing two channels) were obtained more frequently in comparison to control; (b) the unitary conductance of the channel was the same; (c) single-channel openings persisted at high stimulation rates. 6. In contrast to hyperthyroid ventricular cells, I(t) in atrial cells from the same hearts was not substantially changed. 7. The rate dependence of I(t) in atrial cells was also unaffected by hyperthyroidism, in contrast to the large changes observed in ventricular cells. Thus, in atrial cells from hyperthyroid hearts the current was totally suppressed at rates of 1-2 Hz, as in euthyroid conditions. 8. Single-channel recordings in the cell-attached mode showed a unitary conductance similar to that found in normal atrial cells. Channel activity was suppressed at 2 Hz, in contrast to hyperthyroid ventricular cells. 9. In conclusion, I(t) is drastically changed in hyperthyroid rabbit ventricle cells. The changes are in the magnitude of the macroscopic current and its rate dependence. Since the unitary conductance is unchanged (and the peak open probabilities are normally high at positive membrane potential(s) the number of active channels in the membrane must be increased. In atrial cells from the same hyperthyroid hearts no changes are apparent.(ABSTRACT TRUNCATED AT 400 WORDS)

Action Potentials↗

PubMed1992

Beta-adrenergic receptor levels and function after growth of S49 lymphoma cells in low concentrations of epinephrine.

Growth of S49 wild-type (WT) lymphoma cells for 24 hr with 3 nM epinephrine produces a very pronounced attenuation of cAMP accumulation in response to subsequent challenges with much higher concentrations of the catecholamine [Mol. Pharmacol. 36:459-464 (1989)]. We report here the effects of this treatment, in S49 WT, cyc-, and kin- cells, on the responsiveness of adenylate cyclase in partially purified membranes. The desensitization of adenylate cyclase in the S49 WT cells after 24-hr treatment was homologous, in that only responses to epinephrine were attenuated. The EC50 for epinephrine stimulation of adenylate cyclase was 54 +/- 8% (mean +/- standard error) higher in treated cells than in controls, and there was a 32 +/- 3% reduction in Vmax at supramaximal epinephrine concentrations. The treatment also caused a 34 +/- 9% reduction in the levels of the beta-adrenergic receptor (beta AR), which was of a sufficient magnitude to account for the homologous desensitization seen. The 24-hr treatment had similar effects in S49 kin- cells, where we observed a 28 +/- 4% decrease in Vmax, a 35 +/- 6% increase in EC50 for epinephrine stimulation of adenylate cyclase, and a 25 +/- 3% decrease in beta AR. In contrast, the 24-hr treatment had no measurable effect on adenylate cyclase activity in S49 cyc- cells. That is, the responsivity of adenylate cyclase reconstituted with Gs from S49 WT cells was not attenuated, although beta AR levels were significantly decreased. The desensitization of S49 cells with the 24-hr treatment was additive with that mediated by the cAMP-dependent protein kinase (cAPK). Further, unlike the cAPK-mediated attenuation, it was relatively insensitive to the levels of free Mg2+ in the adenylate cyclase reaction mixture. The characteristics of the desensitization produced by 24-hr treatment with 3 nM epinephrine, together with the observation that it is similar in S49 WT and kin- cells, demonstrates that the process in WT cells is, at least in part, independent of the rapid cAPK-mediated desensitization. It is also most likely that it is unrelated to the rapid cAMP-independent processes involving sequestration/internalization or the beta AR kinase, because those mechanisms require much higher receptor occupancies than the 0.2% occurring with 3 nM epinephrine. Thus, 24-hr treatment appears to produce attenuation of adenylate cyclase by causing down-regulation of beta AR, without involving any other known form of desensitization.

Adenylyl Cyclases↗

PubMed1991

T cell non-MHC-restricted antigen-binding molecules secreted or associated with the cell membrane are antigenically distinct.

Some T cells produce membrane-associated or soluble molecules which bind nominal antigen specifically (TABM) and effect immunoregulation or events similar to cell-mediated hypersensitivity. We have used polyclonal antisera raised against an azobenzene arsonate (ABA)-specific TABM secreted by an ABA-specific T cell hybrid or against TNP-specific polypeptides produced by immunoregulatory T cells to identify the expression of soluble (secreted) or membrane-associated TABM. Ascites fluid or culture medium containing a T cell hybrid or T cell lines, respectively, contain TABM recognized only by an antiserum specific for the secreted T cell hybrid (ABA-specific) derived TABM. Conversely, an antiserum that recognized the TNP-specific polypeptides detected cell-membrane associated TABM but did not bind TABM secreted by the T cell hybrid or cell lines.

Animals↗

PubMed1991

Identification of encephalitogenic V beta-4-bearing T cells in SJL mice. Further evidence for the V region disease hypothesis?

Experimental allergic encephalomyelitis (EAE) is an autoimmune disease of the central nervous system mediated by T cells bearing TCR of restricted heterogeneity. Thus, in the murine PL strain, V beta-8.2 is used by 80% of the encephalitogenic T cells. This observation has led to the successful prevention and reversal of EAE by the in vivo use of mAb directed to these restricted gene products. In SJL mice, the V beta-17a gene product has been shown to be used by approximately 50% of encephalitogenic T cells subsequent to immunization with a myelin basic protein (MBP)-derived peptide. However, the other V beta genes used by encephalitogenic T cells in SJL EAE have remained uncharacterized. We now report, for the first time, the beta-chain-encoding DNA sequence of two encephalitogenic, MBP-reactive, SJL-derived T cell clones. These clones which are specific for H-2s and the carboxyl-terminus (amino acid 92-103) of MBP, use TCR encoded by V beta-4. In addition, we demonstrate that the transfer of EAE by a heterogenous SJL-derived encephalitogenic T cell line can be prevented using an anti-V beta-4 antibody in vivo. V beta-4 usage has been previously described in a H-2u/MBP amino-terminus-reactive encephalitogenic T cell. The present findings may thus further support the "V region-disease" hypothesis.

Animals↗