Ribonuclease inhibits Kaposi's sarcoma.
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Antineoplastic Agents↗
Biomedical subjects
D J Adams
Publications and source records attributed to D J Adams.
Co-purification of a ribonuclease and human chorionic gonadotrophin beta-core protein from human urine and displacement of 125I-human luteinizing hormone from Candida albicans binding sites by ribonucleases.
An 18 kDa pregnancy urine protein preparation, purified to apparent electrophoretic homogeneity as judged by silver-staining of polyacrylamide gels, inhibited binding of 125I-hLH (human luteinizing hormone) to Candida albicans microsomes, reacted with monoclonal and polyclonal antibodies raised against human chorionic gonadotrophin (hCG) beta-core protein and exhibited ribonuclease (RNase) activity. Eleven of the 12 amino acids at the N-terminus of a protein in this preparation were identical to those of the N-terminus of human non-secretory ribonuclease. These results indicate co-purification of hCG beta-core with a RNase. An 18 kDa RNase was also purified from a commercial hCG preparation (Chorulon). However, no RNase activity was detected in a highly purified commercial preparation (Profasi). Three commercial RNase preparations displaced 125I-hLH from C. albicans binders at extremely low concentrations (< 0.001 microg/ml RNase) whereas only slight displacement of 125I-hLH from sheep luteal binding sites was observed with very high concentrations of the RNases (100 microg/ml RNase). The co-purification of hCG beta-core and RNase from pregnancy urine and the displacement of 125I-hLH from C. albicans binding sites by RNases may be related to the close relationship that has been identified between mammalian RNase inhibitors and the extracellular domain of gonadotrophin receptors. The presence of RNase in commercial preparations of gonadotrophins should be borne in mind during any investigations that involve impure preparations of these hormones.
Amino Acid Sequence↗
Heterogeneity of nicotinic receptor class and subunit mRNA expression among individual parasympathetic neurons from rat intracardiac ganglia.
Neurons have the potential to form thousands of distinct neuronal nicotinic receptors from the eight alpha and three beta subunits that currently are known. In an effort to determine how much of this potential complexity is realized among individual neurons, we examined the nicotinic pharmacological and biophysical properties and receptor subunit mRNA expression patterns in individual neurons cultured from rat epicardial ganglia. Analysis of the whole-cell pharmacology of these neurons showed a diversity of responses to the agonists acetylcholine, nicotine, cytisine, and 1,1-dimethyl-4-phenylpiperazinium, suggesting that a heterogeneous population of nicotinic receptor classes, or subtypes, is expressed by individual neurons. Single-channel analysis demonstrated three distinct conductances (18, 24, and 31 pS), with patches from different neurons containing different combinations of these channel classes. We used single-cell RT-PCR to examine nicotinic acetylcholine receptor (nAChR) subunit mRNA expression by individual neurons. Although mRNAs encoding all eight neuronal nAChR subunits for which we probed (alpha 2-alpha 5, alpha 7, beta 2-beta 4) were present in multicellular cultures, we found that individual epicardial neurons express distinct subsets of these nAChR subunit mRNAs. These results suggest that individual epicardial neurons express distinct arrays of nAChR subunits and that these subunits may assemble into functional receptors with distinct and variable subunit composition. This variable receptor subunit expression provides an explanation for the diversity of pharmacological and single-channel responses we have observed in individual neurons.
Action Potentials↗
Testing the daily stress stimulus theory of bone adaptation with natural and experimentally controlled strain histories.
Theories of bone adaptation generally consider that a departure in some feature of the normal homeostatic mechanical stimulus governs mechanical adaptation. Specifically, the 'daily stress stimulus' theory commonly used in computational models of bone adaptation suggests that the mechanical stimulus arises from a synthesis of the peak magnitudes from each loading event during a day. In this study, the homeostatic daily strain history of the adult turkey ulna was established by categorizing and counting the natural wing activities of adult male turkeys over a full 24h period. Strain signals were recorded in vivo for each activity type at three mid-diaphysis sites using stacked rosette strain gages. Following surgical isolation and transverse metaphyseal pinning of the ulnae, additional strain signals were recorded during controlled axial and torsional loading regimens associated with documented maintenance, loss, or addition of bone mass. When the present data were incorporated into the daily stress stimulus formulation, the theory did not consistently discriminate maintenance versus formation regimens, i.e., some maintenance regimens were associated with a substantially higher daily stimulus than some regimens causing bone formation.
Adaptation, Physiological↗
Evidence of a role for a guanine nucleotide binding protein during growth and morphogenesis in Candida albicans.
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Blood↗
Characterization of ATP-sensitive potassium channels in freshly dissociated rabbit aortic endothelial cells.
ATP-sensitive potassium (KATP) channels represent a class of K+ channel regulated by intracellular ATP and serve to transduce changes in cell metabolism into changes in membrane potential. The presence of an KATP conductance has recently been demonstrated in freshly dissociated endothelial cells from rabbit arteries. In the present study, the single-channel activity underlying the KATP conductance in rabbit aortic endothelial cells was examined. Unitary currents were evoked in response to lowering intracellular ATP concentration or application of the K(+)-channel activator levcromakalim and were inhibited by the sulfonylurea drug glibenclamide. Exposure of the cytoplasmic face of an inside-out membrane patch to a solution containing 0.1 mM ATP produced single-channel events with unitary conductances of approximately 150 and approximately 25 pS that were inhibited by either 6 mM ATP or 10 microM glibenclamide. A small conductance channel was also activated in cell-attached patches by bath-applied levcromakalim (25 microM). Activation of endothelial cell KATP channels, and subsequent membrane hyperpolarization, may contribute to endothelium-dependent regulation of vascular smooth muscle tone in response to changes in levels of intracellular metabolites.
Adenosine Triphosphate↗
Passive and active membrane properties of isolated rat intracardiac neurons: regulation by H- and M-currents.
The electrical characteristics of isolated neonatal rat intracardiac neurons were examined at 22 and 37 degrees C using the perforated-patch whole cell recording technique. The mean resting membrane potential was -52.0 mV at 37 degrees C and exhibited no temperature dependence. Lowering the temperature from 37 to 22 degrees C decreased the mean input resistance from 854 to 345 Momega, respectively, and reduced the membrane time constant approximately threefold yielding a Q10 of 2.1. Hyperpolarizing current pulses induced time-dependent rectification of the voltage response in all neurons at both temperatures. This behavior was previously not observed in dialyzed neurons and was reversibly blocked by external Cs+ (2 mM) but not Ba2+ (1 mM). Voltage-clamp studies of isolated neurons revealed a hyperpolarization-activated inward current. This inwardly rectifying conductance was isolated from other membrane currents using external Cs+. The time and voltage dependence of this current is consistent with Ih and contributes to the passive electrical properties of rat intracardiac neurons. In >90% of the neurons studied, depolarizing currents evoked firing of multiple, adapting, action potentials at 22 degrees C. The number of action potentials increased with current strength producing a mean discharge of 5.1 (+100 pA, 1 s pulse), which was attenuated at 37 degrees C to a mean of 1.4. The amplitude and kinetics of the slow, muscarine-sensitive inward and outward currents (IM) were highly temperature dependent. Lowering the temperature from 37 to 22 degrees C reduced the steady-state current amplitude by approximately one-third and the rate of deactivation of IM by six- to ninefold at all voltages examined. The average Q10 for the time constant of deactivation of IM was 3.7 +/- 0.3 (mean +/- SE). Acetylcholine (ACh) induced tonic discharges in response to depolarizing currents (+100 pA, 1 s pulse) at both temperatures. This effect of ACh was inhibited by the muscarinic receptor antagonists, pirenzepine (100 nM), and mL-toxin (60 nM). At 37 degrees C, a mean discharge of 1.5 was increased to 23.5 in the presence of ACh. A similar switch from phasic to tonic discharge was also produced by the potassium channel inhibitors, Ba2+ (1 mM) and uridine-5'-triphosphate (UTP; 100 microM), whereas cadmium, 4-aminopyridine, apamin, charybdotoxin, and dendrotoxin did not alter discharge activity. The pharmacological sensitivity profile and temperature dependence of the active membrane properties are consistent with the muscarine-sensitive potassium current (IM) regulating the discharge activity in rat intracardiac neurons.
Acetylcholine↗
M4 muscarinic receptor activation modulates calcium channel currents in rat intracardiac neurons.
Modulation of high-voltage-activated Ca2+ channels by muscarinic receptor agonists was investigated in isolated parasympathetic neurons of neonatal rat intracardiac ganglia using the amphotericin B perforated-patch whole cell recording configuration of the patch-clamp technique. Focal application of the muscarinic agonists acetylcholine (ACh), muscarine, and oxotremorine-M to the voltage-clamped soma membrane reversibly depressed peak Ca2+ channel current amplitude. The dose-response relationship obtained for ACh-induced inhibition of Ba2+ current (IBa) exhibited a half-maximal inhibition at 6 nM. Maximal inhibition of IBa amplitude obtained with 100 microM ACh was approximately 75% compared with control at +10 mV. Muscarinic agonist-induced attenuation of Ca2+ channel currents was inhibited by the muscarinic receptor antagonists pirenzepine (</=300 nM) and m4-toxin (</=100 nM), but not by AF-DX 116 (300 nM) or m1-toxin (60 nM). The dose-response relationship obtained for antagonism of muscarine-induced inhibition of IBa by m4-toxin gave an IC50 of 11 nM. These results suggest that muscarinic agonist-induced inhibition of high-voltage-activated Ca2+ channels in rat intracardiac neurons is mediated by the M4 muscarinic receptor. M4 receptor activation shifted the voltage dependence and depressed maximal activation of Ca2+ channels but had no effect on the steady-state inactivation of Ca2+ channels. Peak Ca2+ channel tail current amplitude was reduced >/=30% at +90 mV in the presence of ACh, indicating a voltage-independent component to the muscarinic receptor-mediated inhibition. Both dihydropyridine- and omega-conotoxin GVIA-sensitive and -insensitive Ca2+ channels were inhibited by ACh, suggesting that the M4 muscarinic receptor is coupled to multiple Ca2+ channel subtypes in these neurons. Inhibition of IBa amplitude by muscarinic agonists was also observed after cell dialysis using the conventional whole cell recording configuration. However, internal perfusion of the cell with 100 microM guanosine 5'-O-(2-thiodiphosphate) trilithium salt (GDP-beta-S) or incubation of the neurons in Pertussis toxin (PTX) abolished the modulation of IBa by muscarinic receptor agonists, suggesting the involvement of a PTX-sensitive G-protein in the signal transduction pathway. Given that ACh is the principal neurotransmitter mediating vagal innervation of the heart, the presence of this inhibitory mechanism in postganglionic intracardiac neurons suggests that it may serve for negative feedback regulation.
Acetylcholine↗
Vasoactive intestinal polypeptide modulation of nicotinic ACh receptor channels in rat intracardiac neurones.
1. The effects of vasoactive intestinal polypeptide (VIP) on isolated parasympathetic neurones of rat intracardiac ganglia were examined under voltage clamp using dialysed and perforated patch whole-cell and excised outside-out membrane patch recording configurations. 2. VIP reversibly potentiated nicotinic ACh-evoked whole-cell currents, with half-maximal potentiation (EC50) obtained with 260 pM VIP. However, VIP had no effect on muscarinic ACh-evoked currents, ATP-evoked currents, or depolarization-activated ionic currents in these neurones. 3. VIP-induced potentiation of nicotinic ACh-evoked whole-cell currents was observed following cell dialysis, and was inhibited reversibly by bath application of the VIP receptor-binding inhibitor L-8-K (5 microM) or the neuronal nicotinic receptor antagonist mecamylamine (3 microM). 4. The signal transduction pathway mediating VIP-induced potentiation of nicotinic ACh-evoked currents involves a guanine nucleotide-binding protein (G-protein) but not cyclic AMP. Intracellular application of 100 microM GDP-beta-S, or pre-incubation of neurones with pertussis toxin, inhibited VIP-induced potentiation of ACh-evoked whole-cell currents. 5. In outside-out membrane patches, co-application of ACh (4 microM) and VIP (4 nM) decreased the duration of closings between bursts and clusters of bursts of ACh single-channel activity relative to control (4 microM, ACh alone). VIP, however, did not alter single ACh receptor channel current amplitude, duration of closings and openings within a burst, or mean burst duration. 6. VIP-induced modification of nicotinic ACh receptor channel kinetics results in an increase in the open-channel probability which is sufficient to account for the VIP-mediated potentiation of nicotinic ACh-evoked whole-cell currents. 7. The potentiation of nicotinic ACh-evoked currents by VIP is likely to account for the altered neuronal activity observed in the mammalian intracardiac ganglia in vivo and consequent changes in heart rate and cardiac contractility.
Acetylcholine↗
Impaired proliferation and tumorigenicity induced by CCAAT/enhancer-binding protein.
A plasmid containing the CCAAT/enhancer-binding protein (C/EBP alpha) gene transcriptionally controlled by the metallothionein promoter was constructed. The gene was transfected into the human hepatocellular carcinoma cell lines Hep3B and HepG2. When cultured in vitro in the presence of 100 microM ZnSO(4), C/EBP alpha expression caused reversible growth arrest. In soft agar clonogenic assays, C/EBP alpha expression decreased both the colony size and the total number of colonies compared with zinc-free controls. C/EBP alpha expressing cells s.c. implanted in CD-1 nu/nu mice were essentially nontumorigenic, whereas C/EBP alpha tumor cells implanted into immunodeficient SCID mice demonstrated a significantly delayed time of tumor appearance compared with cells transfected with a vector control plasmid. These studies suggest that the expression of endogenous genes normally associated with a quiescent, differentiated state, such as C/EBP alpha, can result in impaired proliferative activity and suppressed tumorigenicity of hepatoma cell lines.
Animals↗
An ATP-sensitive potassium conductance in rabbit arterial endothelial cells.
1. Whole-cell patch clamp recording was used to study an ATP-sensitive, sulphonylurea-inhibitable potassium (K+) conductance in freshly dissociated endothelial cells from rabbit arteries. 2. The ATP-sensitive K+ conductance was activated by micromolar concentrations of the K+ channel opener, levcromakalim, and by metabolic inhibition of endothelial cells using dinitrophenol and iodoacetic acid. The current-voltage (I-V) relationship obtained in isotonic K+ solutions was linear between -150 and -50 mV and had a slope conductance of approximately 1 nS. 3. The permeability of the ATP-sensitive K+ conductance determined from reversal potential measurements exhibited the following ionic selectivity sequence: Rb+ > K+ > Cs+ >> Na+ > NH4+ > Li+. 4. Membrane currents activated by either levcromakalim or metabolic inhibition were inhibited by the sulphonylurea drugs, glibenclamide and tolbutamide, with half-maximal inhibitory concentrations of 43 nM and 224 microM and Hill coefficients of 1.1 and 1.2, respectively. Levcromakalim-induced currents were also inhibited by millimolar concentrations of Ba2+ or tetraethylammonium ions in the external solution. 5. Levcromakalim (3 microM) and metabolic inhibition hyperpolarized endothelial cells by approximately 10-15 mV in normal physiological salt solutions. The hyperpolarization induced by levcromakalim or metabolic inhibition was inhibited by bath application of 10 microM glibenclamide. 6. Internal perfusion of the cytosol of whole-cell voltage-clamped endothelial cells with an ATP-free pipette solution activated a membrane current which was reversibly inhibited by internal perfusion with a 3 mM MgATP pipette solution. This current was insensitive to other adenine and guanine nucleotides in the pipette solution. The inward current evoked in a nominally ATP-free internal solution was further increased by bath application of levcromakalim. 7. Levcromakalim (25 microM) did not induce a change in the intracellular Ca2+ concentration of fura-2-loaded endothelial cells, whereas metabolic inhibition caused a slow and sustained increase in intracellular Ca2+ concentration, which was attenuated by 10 microM glibenclamide applied externally.(ABSTRACT TRUNCATED AT 400 WORDS)
Adenosine Triphosphatases↗
Three-dimensional geometric and structural symmetry of the turkey ulna.
Structural models of long-bone preparations usually assume left-right symmetry of contralateral bones under normal (baseline) conditions. To obtain insight on how this assumption affects the detection of subtle changes (as from functional adaptation), we formally examined the three-dimensional geometric and structural symmetry of paired long bones, using contemporary image reconstruction and stress analysis techniques. Nine pairs of ulnae from normal male turkeys were reconstructed computationally from serial transverse images obtained by either (a) mechanical sectioning and digital photographic imaging or (b) computed tomography. Computed tomography scans allowed greater precision in reconstruction than did digitally imaged photographs. Left-right comparisons of parameters of geometric symmetry (from computed tomography reconstructions) revealed average differences in whole bone volume and whole bone principal moments of inertia of 3.6 and 3.0%, respectively. Differences in bone curvature were indexed as noncolinearity of left compared with (mirrored) right centroidal axes, giving a disparity of 0.7 +/- 0.3 mm. Within the longitudinal central 20% of the diaphysis (the customary region for histomorphometry), average left-right differences in cross-sectional area and area principal moments of inertia for computed tomography images were 4.7 and 5.0%, respectively. The overlap of longitudinally paired cross sections of the mid-diaphysis, aligned at common centroids and oriented in the respective principal inertial directions, was greatest (as much as 95%) in the central 20% of the diaphysis. Paired three-dimensional finite element models demonstrated nearly identical left and right stress/strain fields throughout the ulnar diaphyses for both compressive and torsional loading. Our data suggest that the assumption of contralateral geometric symmetry in long bones should be judged in the context of the specific attribute of symmetry under consideration; however, we conclude that for purposes of finite element modeling the assumption of symmetry is reasonable.
Adaptation, Physiological↗
P-glycoprotein mediated resistance to 5'-nor-anhydro-vinblastine (Navelbine).
Navelbine (NVB, vinorelbine tartrate) is a semisynthetic Vinca alkaloid in which the catharanthine moiety contains an eight-membered ring in place of the nine-membered ring that is present in all naturally occurring members of the vinblastine group. This modification selectively reduces interaction with anoxal vs mititotic microtubules and may account for the lower neurotoxicity with improved antitumor activity that has been observed in clinical trials with breast, lung and ovarian cancer. We were interested in whether the structural modification in NVB would also alter the drug resistance profile. Specifically, our aim was to determine whether NVB, like vinblastine (VBL), participates in P-glycoprotein (P-gp)-mediated multidrug resistance (MDR). NVB-resistant, murine P388 cells (P388/NVB), were derived in vivo and used in conjunction with a battery of drug-resistant P388 cell lines in vivo and murine and human tumor cell lines in vitro to develop a resistance profile for NVB. P388/NVB bells were cross-resistant to drugs involved in MDR (doxorubicin, etoposide, amsacrine, vinblastine, vincristine and actinomycin D), but not to the alkylating agents, cyclophosphamide, carmustine, and cisplatin, or to the antimetabolites, 5-fluorouracil and methotrexate. P388/NVB cellular resistance to NVB was stable without drug pressure during continuous passage in vivo for more than ten weeks and in vitro for at least five weeks. These cells exhibited increased expression of P-gp, and a 30-fold level of resistance of NVB in vitro, which was completely reversable with verapamil. The MDR phenotype was confirmed in other tumor models. P388 tumors resistant to vinblastine, vincristine, doxorubicin, and etoposide were cross-resistant to NVB in vivo.(ABSTRACT TRUNCATED AT 250 WORDS)
ATP Binding Cassette Transporter, Subfamily B, Mem↗
The relationship of brevetoxin 'length' and A-ring functionality to binding and activity in neuronal sodium channels.
BACKGROUND: Brevetoxins are polyether ladder toxins that are ichthyotoxic at nanomolar concentrations. They bind to voltage-gated sodium channels, causing four distinct electrophysiological effects: (i) a shift of activation potential; (ii) occurrence of subconductance states; (iii) induction of longer mean open times of the channel; and (iv) inhibition of channel inactivation. We set out to determine whether these functions all require the same structural elements within the brevetoxin molecules. RESULTS: Several synthetically prepared structural analogs of brevetoxin B were examined in synaptosome receptor binding assays and by functional electrophysiological measurements. A truncated analog is not ichthyotoxic at micromolar concentrations, shows decreased receptor-binding affinity, and causes only a shift of activation potential without affecting mean open times or channel inactivation. An analog with the A-ring carbonyl removed binds to the receptor with nanomolar affinity, produces a shift of activation potential and inhibits inactivation, but does not induce longer mean open times. An analog in which the A-ring diol is reduced shows low binding affinity, yet populates five subconductance states. CONCLUSIONS: Our data are consistent with the hypothesis that binding to sodium channels requires an elongated cigar-shaped molecule, approximately 30 A long. The four electrophysiological effects of the brevetoxins are not produced by a single structural feature, however, since they can be decoupled by using modified ligands, which are shown here to be partial sodium channel agonists. We propose a detailed model for the binding of brevetoxins to the channel which explains the differences in the effects of the brevetoxin analogs. These studies also offer the potential for developing brevetoxin antagonists.
Animals↗
Monovalent and divalent cation permeability and block of neuronal nicotinic receptor channels in rat parasympathetic ganglia.
Acetylcholine-evoked currents mediated by activation of nicotinic receptors in rat parasympathetic neurons were examined using whole-cell voltage clamp. The relative permeability of the neuronal nicotinic acetylcholine (nACh) receptor channel to monovalent and divalent inorganic and organic cations was determined from reversal potential measurements. The channel exhibited weak selectivity among the alkali metals with a selectivity sequence of Cs+ > K+ > Rb+ > Na+ > Li+, and permeability ratios relative to Na+ (Px/PNa) ranging from 1.27 to 0.75. The selectivity of the alkaline earths was also weak, with the sequence of Mg2+ > Sr2+ > Ba2+ > Ca2+, and relative permeabilities of 1.10 to 0.65. The relative Ca2+ permeability (PCa/PNa) of the neuronal nACh receptor channel is approximately fivefold higher than that of the motor endplate channel (Adams, D. J., T. M. Dwyer, and B. Hille. 1980. Journal of General Physiology. 75:493-510). The transition metal cation, Mn2+ was permeant (Px/PNa = 0.67), whereas Ni2+, Zn2+, and Cd2+ blocked ACh-evoked currents with half-maximal inhibition (IC50) occurring at approximately 500 microM, 5 microM and 1 mM, respectively. In contrast to the muscle endplate AChR channel, that at least 56 organic cations which are permeable to (Dwyer et al., 1980), the majority of organic cations tested were found to completely inhibit ACh-evoked currents in rat parasympathetic neurons. Concentration-response curves for guanidinium, ethylammonium, diethanolammonium and arginine inhibition of ACh-evoked currents yielded IC50's of approximately 2.5-6.0 mM. The organic cations, hydrazinium, methylammonium, ethanolammonium and Tris, were measureably permeant, and permeability ratios varied inversely with the molecular size of the cation. Modeling suggests that the pore has a minimum diameter of 7.6 A. Thus, there are substantial differences in ion permeation and block between the nACh receptor channels of mammalian parasympathetic neurons and amphibian skeletal muscle which represent functional consequences of differences in the primary structure of the subunits of the ACh receptor channel.
Acetylcholine↗
Assay of vancomycin by fluorescence polarisation immunoassay and EMIT in patients with renal failure.
Serum with vancomycin concentrations between 5 and 15 mg/L from patients on dialysis were assayed by fluorescence polarization immunoassay (FPIA) and enzyme multiplied immunoassay technique (EMIT). The concentrations as determined by FPIA were higher than those using EMIT (mean difference 2.1 mg/L, S.D. 1.5; range -0.8-6.3) with substantial interpatient variability in the difference between the two assays. This suggests that concentrations of active vancomycin are lower than indicated by FPIA and thresholds for redosing may need to be adjusted.
Enzyme Multiplied Immunoassay Technique↗
A prospective laboratory-based audit of gentamicin use and therapeutic monitoring.
We report a study investigating the proportion of patients in whom therapeutic serum concentrations of gentamicin are achieved in the early phase of treatment and to determine the underlying reasons for sub-optimal therapy. A laboratory based prospective study of 83 courses of gentamicin was performed (excluding patients receiving renal replacement therapy or with bacterial endocarditis) in a London teaching hospital. Of 83 monitored courses 74 had paired levels tested. Initial trough concentrations were > 2 mg/L in nine (12%) and were < 1 mg/L in 51 (69%) indicating levels were seldom in the toxic range. The first monitored peaks were sub-therapeutic in 58 (78%) courses using a cut-off of 5 mg/L and only seven (9%) were greater than 6 mg/L. Of these seven patients, six had troughs levels of greater than 2 mg/L. Of those with initial levels below 5 mg/L, 33 had further serum levels tested, of which 26 (79%) remained below 5 mg/L. Of those for whom dosing information was available 73% received 80 mg t.d.s. and the mean dose for those for which body weight was known was 3.3 mg/kg/day (S.D. = 0.7). Most patients continued to receive 8-hourly dosing with 80 mg of gentamicin, leading to subtherapeutic peak levels in the majority of cases. In those patients with satisfactory peaks, such dosing frequently leads to elevated troughs. This suggests that such dosing practices should be abandoned and replaced with dosing based on body weight and divided into no more than two daily doses.
Anti-Bacterial Agents↗
Caffeine-evoked, calcium-sensitive membrane currents in rabbit aortic endothelial cells.
1. Single cell photometry and whole-cell patch clamp recording were used to study caffeine-induced intracellular Ca2+ signals and membrane currents, respectively, in endothelial cells freshly dissociated from rabbit aorta. 2. Caffeine (5 mM) evoked a transient increase in [Ca2+]i in fura-2-loaded endothelial cells. Pretreatment of cells with 10 microM ryanodine did not alter resting [Ca2+]i but irreversibly inhibited the caffeine-induced rise in [Ca2+]i. The caffeine-induced increase in [Ca2+]i was not attenuated by the removal of extracellular Ca2+ and did not stimulate the rate of Mn2+ quench of fura-2 fluorescence. 3. Bath application of caffeine evoked a dose- and voltage-dependent outward current. The rate of onset and amplitude of the caffeine-evoked outward current increased with higher caffeine concentrations and membrane depolarization. The relationship between caffeine-evoked current amplitude and membrane potential was non linear, suggesting that the channels underlying the current are voltage-sensitive. 4. In the absence of extracellular Ca2+, the amplitude of the caffeine-evoked outward current was reduced by approximately 50% but the duration of the current was prolonged compared to that observed in the presence of external Ca2+. Ca(2+)-free external solutions produced an unexpected increase in both the frequency and amplitude of spontaneous transient outward currents (STOCs). 5. Inclusion of heparin (10 micrograms ml-1) in the patch pipette abolished the acetylcholine (ACh)-induced outward current but failed to inhibit either STOCs or the caffeine-evoked outward current in native endothelial cells. In the absence of extracellular Ca2+, heparin did not affect either STOCs or the caffeine-induced outward current. 6. Externally applied tetraethylammonium ions (TEA, 3-10mM) reversibly inhibited unitary Ca2+-activated K+ currents and STOCs in endothelial cells but failed to inhibit completely the outward current evoked by 20 mM caffeine.7. Bath application of 0.1 mM zinc ion (Zn2+), a chloride channel blocker, did not affect unitary currents or STOCs but reduced the amplitude of the caffeine-evoked current by >75% compared to control. Replacement of extracellular NaCl with Na gluconate also reduced the amplitude of the caffeine-induced outward current. Bath application of 0.1 mM Zn2+ and 10 mM TEA completely blocked the caffeine-evoked outward current in endothelial cells.8. Caffeine-induced Ca2+ release from intracellular stores evokes a transient rise in [Ca2+1, which is correlated with a large, transient outward current. The ionic dependence and inhibition of the caffeine sensitive current by TEA and Zn2+ suggests that Ca2+-activated K+ and Cl- conductances contribute to the caffeine response in rabbit aortic endothelial cells.
Animals↗