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Biomedical subjects

R Horn

Publications and source records attributed to R Horn.

At least 73 records · Page 4Linked to original sources
PubMed1995

A molecular link between activation and inactivation of sodium channels.

A pair of tyrosine residues, located on the cytoplasmic linker between the third and fourth domains of human heart sodium channels, plays a critical role in the kinetics and voltage dependence of inactivation. Substitution of these residues by glutamine (Y1494Y1495/QQ), but not phenylalanine, nearly eliminates the voltage dependence of the inactivation time constant measured from the decay of macroscopic current after a depolarization. The voltage dependence of steady state inactivation and recovery from inactivation is also decreased in YY/QQ channels. A characteristic feature of the coupling between activation and inactivation in sodium channels is a delay in development of inactivation after a depolarization. Such a delay is seen in wild-type but is abbreviated in YY/QQ channels at -30 mV. The macroscopic kinetics of activation are faster and less voltage dependent in the mutant at voltages more negative than -20 mV. Deactivation kinetics, by contrast, are not significantly different between mutant and wild-type channels at voltages more negative than -70 mV. Single-channel measurements show that the latencies for a channel to open after a depolarization are shorter and less voltage dependent in YY/QQ than in wild-type channels; however the peak open probability is not significantly affected in YY/QQ channels. These data demonstrate that rate constants involved in both activation and inactivation are altered in YY/QQ channels. These tyrosines are required for a normal coupling between activation voltage sensors and the inactivation gate. This coupling insures that the macroscopic inactivation rate is slow at negative voltages and accelerated at more positive voltages. Disruption of the coupling in YY/QQ alters the microscopic rates of both activation and inactivation.

Base Sequence↗
PubMed1995

Abnormal spermatozoa in the cauda epididymidis of adult and aged hamsters (Mesocricetus auratus): a study by electron microscopy.

The morphology of the spermatozoa in the cauda epididymidis of 6- and 24-month-old (adult and aged) hamster was studied by electron microscopy. Lesions found in the spermatozoa included alterations in the acrosomal matrix and nuclear membranes, abnormal or absent mitochondria, alterations in the axonemes, dense fibres and microtubules, and bent flagella. All these abnormalities are similar to those found in other species and few alterations were exclusive to the older animals. Ultrastructurally abnormal spermatozoa were observed in both groups but were present at a significantly greater frequency in aged hamsters (p < 0.005). the percentage of sperm with abnormal acrosomes, mitochondria and dense fibres and of bent (but not disrupted) spermatozoa was significantly greater in the older animals. These results show that the ultrastructural alterations in hamster sperm are similar to those found in other mammals. Moreover, the percentage of ultrastructurally abnormal spermatozoa in aged hamsters is greater than 6-month-old hamsters, this increase is not accompanied by any new kinds of alteration.

Aging↗
PubMed1995

Histochemical study of glycoconjugates in the epididymis of the hamster (Mesocricetus auratus).

The glycoconjugates of hamster epididymis were investigated with conventional and lectin histochemistry. A zone of the caput epididymis, with particular histochemical characteristics, has been differentiated. beta-Elimination in combination with lectins was used to establish the presence and distribution of N- and O-linked glycoconjugates. The epithelium, spermatozoa and the intertubular matrix were rich in glycoconjugates. The Golgi apparatus and stereocilia of the principal cells were intensely positive with HPA, PNA and SBA lectins. beta-Elimination indicated that these cells contained abundant O-linked glycoconjugates. Apical and clear cells presented a common lectin affinity; their reactivities towards WGA and UEA-I were very positive. These cells probably contain abundant N-glycoconjugates. The spermatozoa were stained by periodic acid-Schiff (PAS) and by all the lectins (especially in the acrosome), except by those with an affinity for alpha-L-fucosyl residues; the most intense reaction was found with HPA, WGA, PNA and SBA. Changes in the sperm lectin binding along the ductus were observed: sperm flagellum abruptly acquired WGA and PNA labelling from the posterior caput, and HPA reactivity was negative only in the zone between the caput and the corpus.

Animals↗
PubMed1995

Characterizing the mu-conotoxin binding site on voltage-sensitive sodium channels with toxin analogs and channel mutations.

The three-dimensional organization of the domains of the rat skeletal muscle sodium channel subtype 1 (rSkM1) and the toxin-channel interaction surface have been explored by a complementary mutagenesis approach. This method involves probing mutant channels with analogs of the peptide toxin, mu-conotoxin (mu-CTX), for which the tertiary structure has been determined. mu-CTX has an overall net charge of +5. The blocking of Na+ currents of rSkM1 expressed in Xenopus oocytes by mu-CTX analogs in which negative charge had been removed by Asn substitution for Asp or positive charge had been decreased by Gln substitution for Arg or Lys was studied; the mu-CTX analogs exhibited decreased blocking potencies of up to 228-fold compared with an IC50 = 51.4 +/- 2.2 nM for native mu-CTX on wild-type rSkM1. Mutations at Arg 13 of mu-CTX were the most critical in decreasing potency and at Lys9 were the least critical. Charge alone, however, was not the essential factor in some toxin substitutions: the IC50 value for Asp12Asn showed little change while that for Asp12Glu was increased approximately 100-fold due to a change in conformation (revealed by NMR measurements of the toxin in solution). Focusing on the sites in the channel which might be involved in toxin binding, mutations were introduced involving substitutions at more than a dozen mostly anionic sites in putative extracellular residues of rSkM1. The toxin binding results indicate: firstly, many channel mutations at anionic sidechains on the putative extracellular surface of mu-CTX-sensitive channels, thought to be possible sites of interaction with toxin, have been shown to have no effect on toxin binding. Secondly, one channel mutation, rSkM1/Tyr401Cys, (in the loop between S5 and S6 of Domain 1), affected mu-CTX potency causing a 3.7-fold increase in IC50 value. The ratio of toxin blocking potencies was not significantly different when wild-type and the mutant (Tyr401Cys) rSkM1 channels were studied with two toxin analogs, Arg19Gln and Arg13Gln, in contrast to all other toxin derivatives examined. Since Tyr401 is known to be in the channel pore, these results suggest that either or both of the Arg residues at positions 13 and 19 of mu-CTX interact(s) with residue Tyr401 of rSkM1 and, therefore, indicate that mu-CTX extends into the pore region of the channel.

Amino Acid Sequence↗
PubMed1994

Sodium channel mutations in paramyotonia congenita exhibit similar biophysical phenotypes in vitro.

Mutations in the skeletal muscle voltage-gated Na+ channel alpha-subunit have been found in patients with two distinct hereditary disorders of sarcolemmal excitation: hyperkalemic periodic paralysis (HYPP) and paramyotonia congenita (PC). Six of these mutations have been functionally expressed in a heterologous cell line (tsA201 cells) using the recombinant human skeletal muscle Na+ channel alpha-subunit cDNA hSkM1. PC mutants from diverse locations in this subunit (T1313M, L1433R, R1448H, R1448C, A1156T) all exhibit a similar disturbance in channel inactivation characterized by reduced macroscopic rate, accelerated recovery, and altered voltage dependence. PC mutants had no significant abnormality in activation. In contrast, one HYPP mutation studied (T704M) has a normal inactivation rate but exhibits shifts in the midpoints of steady-state activation and inactivation along the voltage axis. These findings help to explain the phenotypic differences between HYPP and PC at the molecular and biophysical level and contribute to our understanding of Na+ channel structure and function.

Base Sequence↗
PubMed1994

Western blot analysis of thyrotropin receptor expression in human thyroid tumours and correlation with TSH-binding.

Expression of thyrotropin receptor (TSHr) was analyzed in normal human thyroids and differentiated thyroid tumours by Northern blotting of total RNA and by Western blotting of detergent-solubilized membrane proteins with an antibody specific for the extracellular domain of TSHr. Under nonreducing conditions TSHr stained as a 90-kDa protein in normal thyroid tissue. Expression varied in differentiated carcinomas from normal to not detectable in parallel to steady state TSHr mRNA levels and TSH binding in a TSH binding assay. The negative expression of TSHr in some patients with differentiated carcinomas may have consequences for the TSH suppressive treatments of these patients.

Binding Sites↗
PubMed1994

Morphological and histochemical study of human submucosal laryngeal glands.

BACKGROUND: The respiratory submucosal glands are a major source of secretions in the airway. Human submucosal laryngeal glands have been scarcely studied, with no works existing about their ultrastructure and histochemistry. METHODS: Samples of epiglottis, ventricle, false vocal folds and true vocal folds were fixed in 10% buffered formalin for histochemical study with conventional and carbohydrate lectin histochemistry. Other samples were fixed in 2.5% glutaraldehyde and conventionally processed for transmission electron microscopy. RESULTS: The human submucosal laryngeal glands are composed of serious tubules; mucous tubules; collector duct; and final portion of this duct. The serous cells showed sialosulphomucins and affinity for WGA and Con-A lectins. With a previous treatment with neuraminidase, they also labelled with PNA. The mucous cells contained sialosulphomucins and showed affinity for WGA and DBA lectins in the samples proceeding from blood group A, and for WGA, UEA-I and LTA with those from blood group O. Ultrastructurally, the serous cells presented a wide variety of granules, cells in which seromucous granules predominated. The mucous cells presented larger-sized granules which were very electron-lucent. The collector duct was composed of mitochondria-rich cells and basal cells. A cell which we have termed "intermediate" was identified in the transition zone between the mucous tubules and the collector duct, and in the final portion of the collector duct. It had morphological characteristics as if it were a transition between a goblet cell and collector duct cell. Some nerve endings with cholinergic and peptidergic vesicles were found among the myoepithelial cells. CONCLUSIONS: These glands presented some histological differences from the bronchial glands, the mucous secretion was related to the blood group antigens, and the serous cells showed a wide variability in their secretory granules, many of them being of a seromucous type.

Adult↗
PubMed1994

Functional expression and properties of the human skeletal muscle sodium channel.

Full-length deoxyribonucleic acid, complementary (cDNA) constructs encoding the alpha-subunit of the adult human skeletal muscle Na+ channel, hSkM1, were prepared. Functional expression was studied by electrophysiological recordings from cRNA-injected Xenopus oocytes and from transiently transfected tsA201 cells. The Na+ currents of hSkM1 had abnormally slow inactivation kinetics in oocytes, but relatively normal kinetics when expressed in the mammalian cell line. The inactivation kinetics of Na+ currents in oocytes, during a depolarization, were fitted by a weighted sum of two decaying exponentials. The time constant of the fast component was comparable to that of the single component observed in mammalian cells. The block of hSkM1 Na+ currents by the extracellular toxins tetrodotoxin (TTX) and mu-conotoxin (microCTX) was measured. The IC50 values were 25 nM (TTX) and 1.2 microM (microCTX) in oocytes. The potency of TTX is similar to that observed for the rat homolog rSkM1, but the potency of microCTX is 22-fold lower in hSkM1, primarily due to a higher rate of toxin dissociation in hSkM1. Single-channel recordings were obtained from outside-out patches of oocytes expressing hSkM1. The single-channel conductance, 24.9 pS, is similar to that observed for rSkM1 expressed in oocytes.

Animals↗
PubMed1994

Sodium channel mutations in paramyotonia congenita uncouple inactivation from activation.

Mutations in the adult human skeletal muscle Na+ channel alpha subunit cause the disease paramyotonia congenita. Two paramyotonia congenita mutations, R1448H and R1448C, substitute histidine and cysteine for arginine in the S4 segment of domain 4. These mutations, expressed in a cell line, have only small effects on the activation of Na+ currents, but mutant channels inactivate more slowly with less voltage dependence than wild-type channels and exhibit an enhanced rate of recovery from inactivation. Increase of extracellular pH made the rate of inactivation of R1448H similar to that of R1448C, suggesting that this residue has an extracellular location and that its charge is important for normal inactivation. Analysis of single-channel data reveals that mutant channels inactivate normally from closed states, but poorly from the open state. The data suggest a critical role for the S4 helix of domain 4 in coupling between activation and inactivation.

Base Sequence↗
PubMed1994

Internal block of human heart sodium channels by symmetrical tetra-alkylammoniums.

The human heart Na channel (hH1) was expressed by transient transfection in tsA201 cells, and we examined the block of Na current by a series of symmetrical tetra-alkylammonium cations: tetramethylammonium (TMA), tetraethylammonium (TEA), tetrapropylammonium (TPrA), tetrabutylammonium (TBA), and tetrapentylammonium (TPeA). Internal TEA and TBA reduce single-channel current amplitudes while having little effect on single channel open times. The reduction in current amplitude is greater at more depolarized membrane potentials. Analysis of the voltage-dependence of single-channel current block indicates that TEA, TPrA and TBA traverse a fraction of 0.39, 0.52, and 0.46 of the membrane electric field to reach their binding sites. Rank potency determined from single-channel experiments indicates that block increases with the lengths of the alkyl side chains (TBA > TPrA > TEA > TMA). Internal TMA, TEA, TPrA, and TBA also reduce whole-cell Na currents in a voltage-dependent fashion with increasing block at more depolarized voltages, consistent with each compound binding to a site at a fractional distance of 0.43 within the membrane electric field. The correspondence between the voltage dependence of the block of single-channel and macroscopic currents indicates that the blockers do not distinguish open from closed channels. In support of this idea TPrA has no effect on deactivation kinetics, and therefore does not interfere with the closing of the activation gates. At concentrations that substantially reduce Na channel currents, TMA, TEA, and TPrA do not alter the rate of macroscopic current inactivation over a wide range of voltages (-50 to +80 mV). Our data suggest that TMA, TEA, and TPrA bind to a common site deep within the pore and block ion transport by a fast-block mechanism without affecting either activation or inactivation. By contrast, internal TBA and TPeA increase the apparent rate of inactivation of macroscopic currents, suggestive of a block with slower kinetics.

Heart↗
PubMed1994

Evidence for a direct interaction between internal tetra-alkylammonium cations and the inactivation gate of cardiac sodium channels.

The effects of internal tetrabutylammonium (TBA) and tetrapentylammonium (TPeA) were studied on human cardiac sodium channels (hH1) expressed in a mammalian tsA201 cell line. Outward currents were measured at positive voltages using a reversed Na gradient. TBA and TPeA cause a concentration-dependent increase in the apparent rate of macroscopic Na current inactivation in response to step depolarizations. At TPeA concentrations < 50 microM the current decay is well fit by a single exponential over a wide voltage range. At higher concentrations a second exponential component is observed, with the fast component being dominant. The blocking and unblocking rate constants of TPeA were estimated from these data, using a three-state kinetic model, and were found to be voltage dependent. The apparent inhibition constant at 0 mV is 9.8 microM, and the blocking site is located 41 +/- 3% of the way into the membrane field from the cytoplasmic side of the channel. Raising the external Na concentration from 10 to 100 mM reduces the TPeA-modified inactivation rates, consistent with a mechanism in which external Na ions displace TPeA from its binding site within the pore. TBA (500 microM) and TPeA (20 microM) induce a use-dependent block of Na channels characterized by a progressive, reversible, decrease in current amplitude in response to trains of depolarizing pulses delivered at 1-s intervals. Tetrapropylammonium (TPrA), a related symmetrical tetra-alkylammonium (TAA), blocks Na currents but does not alter inactivation (O'Leary, M. E., and R. Horn. 1994. Journal of General Physiology. 104:507-522.) or show use dependence. Internal TPrA antagonizes both the TPeA-induced increase in the apparent inactivation rate and the use dependence, suggesting that all TAA compounds share a common binding site in the pore. A channel blocked by TBA or TPeA inactivates at nearly the normal rate, but recovers slowly from inactivation, suggesting that TBA or TPeA in the blocking site can interact directly with a cytoplasmic inactivation gate.

Binding Sites↗
PubMed1994

Voltage-dependent regulation of modal gating in the rat SkM1 sodium channel expressed in Xenopus oocytes.

The TTX-sensitive rat skeletal muscle sodium channel (rSkM1) exhibits two modes of inactivation (fast vs slow) when the alpha subunit is expressed alone in Xenopus oocytes. In this study, two components are found in the voltage dependence of normalized current inactivation, one having a V1/2 in the expected voltage range (approximately -50 mV, I(N)) and the other with a more hyperpolarized V1/2 (approximately -130 mV, IH) at a holding potential of -90 mV. The I(N) component is associated with the gating mode having rapid inactivation and recovery from inactivation of the macroscopic current (N-mode), while IH corresponds to the slow inactivation and recovery mode (H-mode). These two components are interconvertible and their relative contribution to the total current varies with the holding potential: I(N) is favored by hyperpolarization. The interconversion between the two modes is voltage dependent and is well fit to a first-order two-state model with a voltage dependence of e-fold/8.6 mV and a V1/2 of -62 mV. When the rat sodium channel beta 1-subunit is coinjected with rSkM1, IH is essentially eliminated and the inactivation kinetics of macroscopic current becomes rapid. These two current components and their associated gating modes may represent two conformations of the alpha subunit, one of which can be stabilized either by hyperpolarization or by binding of the beta 1 subunit.

Animals↗
PubMed1994

Calcium channels in excitable cells: divergent genotypic and phenotypic expression of alpha 1-subunits.

The Ba2+ currents and mRNA levels of four members of the rat brain family of alpha 1-subunit Ca2+ channel genes were examined and compared in the rat cell lines GH3 and PC-12 and in the mouse lines NIE-115 and AtT-20. The RNA was measured with ribonuclease protection assays using probes derived from rat brain (rb) Ca2+ channel cDNAs (rbA, rbB, rbC, and rbD), and the Ba2+ currents were studied by whole cell patch-clamp recording. L-, N-, P-, and T-type currents were discriminated by the voltage dependence and pharmacological properties of Ba2+ currents. All cell lines expressed all four rat brain Ca2+ channel genes, except GH3 cells, which lacked rbB. The functional diversity of Ba2+ currents, however, was quite different among the cell lines. GH3 cells showed evidence of L- and T-type currents, undifferentiated PC-12 cells of L-type currents, AtT-20 cells of L-, N-, and P-type currents, and undifferentiated NIE-115 cells of a T-type current that was partially blocked by both nifedipine and BAY K 8644. Dimethyl sulfoxide-differentiated NIE-115 cells also had an L-type current. Differentiation of NIE-115 cells caused an increase in the levels of rbB, rbC, and rbD RNAs. Differentiation by nerve growth factor caused an increase in levels of all four genes in PC-12. Our data give further support for the assignment of rbA, rbB, and rbC/rbD gene products as components of P-, N-, and L-type Ca2+ channels, respectively.

3-Pyridinecarboxylic acid, 1,4-dihydro-2,6-dimethy↗
PubMed1994

Pulse amplitude and frequency modulation of parathyroid hormone in early postmenopausal women before and on hormone replacement therapy.

Although the pathophysiology of postmenopausal osteoporosis has been investigated extensively, it is still not established in what respect PTH is related to the events. Recently, consistent data on the pulsatile secretion of PTH in man have been published. In this study intact PTH was measured in six early postmenopausal women before and after 6 months of hormone replacement therapy (HRT; 0.6 mg conjugated estrogens and 5 mg medrogestone). In addition to parameters of calcium metabolism and bone mass and to control HRT, intact PTH was measured in blood drawn over 6 h every 2 min. With HRT there was a 30% reduction in PTH secretion. Both the amount secreted per pulse (baseline, 26.8 +/- 6.9 ng/L; HRT, 21.4 +/- 7.6 ng/L; P < 0.05) as well as the basal secretion (baseline, 232.6 +/- 117.6 ng/L.h; HRT, 145.5 +/- 80.0 ng/L.h; P < 0.01) were reduced, whereas the pulse count per h remained constant (baseline, 5.1 +/- 2.2; HRT, 5.1 +/- 1.3). Power spectrum analysis showed a shift in spectral maxima consistent with these findings. Ionized and total calcium were slightly, but nonsignificantly, reduced with treatment. In summary we conclude that in early postmenopausal women, HRT reduces the secretion of PTH by reducing both the basal secretion and the amount secreted per pulse. It is conceivable that some of the known effects of HRT on bone metabolism might be mediated by the modulation of PTH secretion.

Calcium↗
PubMed1994

Pulse amplitude and frequency modulation of parathyroid hormone in primary hyperparathyroidism.

Pulsatile secretion of PTH in human subjects has been described recently. However, the pattern of PTH secretion in primary hyperparathyroidism (pHPT) remains to be characterized. In this study intact PTH was measured in 9 female patients with pHPT. As a control group we present data from 10 postmenopausal women. In addition to parameters of calcium metabolism and bone mass, PTH was measured in samples drawn over 4 or 6 h every 2 min by central venous blood sampling. The mean intact PTH concentration was 39.0 +/- 20.3 ng/L in healthy women and 193.2 +/- 127.9 ng/L in female patients with pHPT (P < 0.01). Pulse rhythm analysis showed significant differences between both groups for total PTH secretion per h (patients, 1196.4 +/- 485.3 ng/L; control group, 271.7 +/- 132.2 ng/L), basal PTH secretion per h (patients, 852.4 +/- 459.1 ng/L; control group, 185.6 +/- 126.1 ng/L), and average PTH secretion per pulse (patients, 112.6 +/- 54.8 ng/L; control group, 23.2 +/- 7.1 ng/L). Both patients and control subjects had, on an average, five pulses per h, and the pulsatile secretion accounted for about 50% of the total secretion. Differences in power spectrum analysis were consistent with these findings. The cross-correlation of PTH and calcium indicates an impaired feedback regulation in pHPT. PTH secretion in female patients with pHPT results from both an increased basal secretion and an increased amplitude of PTH pulses. Other features of secretion are the same as those in normal women. Feedback regulation of PTH and calcium is impaired in pHPT.

Aged↗
PubMed1994

Managed care: implications for underrepresented physicians.

The advent of health care reform has brought uncertainty to the lives of underrepresented physicians and their patients. Managed care promises increased numbers of primary care physicians and fewer specialists; increased numbers of primary care physicians and fewer specialists; increased numbers of group practices and health maintenance organizations (HMOs); and combinations of health care plans and insurance plans. Solo practice fee-for-service physicians will find it more difficult to compete. Many physicians will retire or change careers; some will be left out of managed care plans altogether. To cope, underrepresented physicians must encourage the enrollment of more minorities in medical schools and residency training programs. They must become involved in the management and administration of managed care programs. They must become involved in politics. Finally, they must combine forces to become competitive in the changing health care market.

Health Care Reform↗