PubMed Health⌕ Search

Biomedical subjects

W M Weber

Publications and source records attributed to W M Weber.

36 records · Page 2Linked to original sources
PubMed1997

Suppression of neuropeptide Y1 receptor function in SK-N-MC cells by nitric oxide.

The neuropeptide Y1 receptor (NPY1) predominantly mediates the vasoconstrictor effects of NPY in smooth muscle cells. The present experiments were planned to study the direct influence of the vasodilator nitric oxide (NO) on NPY1-receptor function. SK-N-MC and CHP-234 cells expressing Y1 and Y2 receptor, respectively, were incubated with the NO donors sodium nitroprusside (SNP), 3-morpholinosydnonimine (SIN-1), and S-nitroso-N-acetyl-penicillamine (SNAP). Receptor binding, Y1-receptor mRNA expression by Northern blot, and adenosine 3',5'-cyclic monophosphate (cAMP) and intracellular Ca2+ concentration ([Ca2+]i) responses were studied. SNP, SIN-1, and SNAP decreased normal binding of NPY to the NPY1 receptor in SK-N-MC cells in a concentration-dependent manner. SNP (500 microM), SIN-1 (1,000 microM), and SNAP (500 microM) significantly decreased binding to approximately 50%. The cell viability was not reduced. None of the NO donors affected Y2 receptor binding. Pretreatment with SNP significantly attenuated NPY-induced inhibition of cAMP formation in SK-N-MC cells but had no effect on CHP cells. The NPY-induced [Ca2+]i response was reduced to 50% by SNP pretreatment. NPY1 mRNA expression was reduced to one-third after SNAP treatment of SK-N-MC cells. In vitro, NPY1 receptor function of SK-N-MC cells is inhibited by NO-donor incubation on an mRNA level.

Blotting, Northern↗
PubMed1996

Structural characterization of a novel neuropeptide from the central nervous system of the leech Erpobdella octoculata. The leech osmoregulator factor.

Purification of a material immunoreactive to an antiserum against the C-terminal part of the oxytocin (Pro-Leu-Gly-amide) and present in the central nervous system of the Pharyngobdellid leech Erpobdella octoculata was performed by reversed-phase high performance liquid chromatography combined with both enzyme-linked immunosorbent and dot immunobinding assays for oxytocin. The amino acid sequence of the purified peptide (Ile-Pro-Glu-Pro-Tyr-Val-Trp-Asp) was established by Edman degradation and confirmed by electrospray mass spectrometry measurement. When injected in leeches, purified or synthetic peptides exert an anti-diuretic effect, the most effective ranged between 10 pmol and 1 nmol. They provoked an uptake of water 1-2 h post-injection. Furthermore, electrophysiological experiments conducted in the leech Hirudo medicinalis revealed an inhibition of the potency of Na+ conductances of leech skin by this peptide. Immunocytochemical studies with an antiserum against synthetic oxytocin-like molecule provided the cytological basis for existence of a neuropeptide, since large amounts of immunoreactive neurons were detected in the central nervous systems of E. octoculata. The purified molecule is both different to peptides of the oxytocin/vasopressin family and is a novel neuropeptide in the animal kingdom. It was named the leech osmoregulator factor (LORF). An identification of the proteins immunoreactive to an antiserum against oxytocin performed at the level of both central nervous systems extracts and in vitro central nervous system-translated RNA products indicated that in the two cases, a single protein was detected. These proteins with a molecular masses of, respectively, approximately 34 kDa (homodimer of 17 kDa) for the central nervous systems extracts and approximately 19 kDa for in vitro central nervous system-translated RNA products were not recognized by the antiserum against MSEL- and VLDV-neurophysin (proteins associated to oxytocin and vasopressin), confirming that LORF did not belong to the oxytocin/vasopressin family.

Amino Acid Sequence↗
PubMed1996

Expression cloning and functional characterization of the kidney cortex high-affinity proton-coupled peptide transporter.

The presence of a proton-coupled electrogenic high-affinity peptide transporter in the apical membrane of tubular cells has been demonstrated by microperfusion studies and by use of brush border membrane vesicles. The transporter mediates tubular uptake of filtered di- and tripeptides and aminocephalosporin antibiotics. We have used expression cloning in Xenopus laevis oocytes for identification and characterization of the renal high-affinity peptide transporter. Injection of poly(A)+ RNA isolated from rabbit kidney cortex into oocytes resulted in expression of a pH-dependent transport activity for the aminocephalosporin antibiotic cefadroxil. After size fractionation of poly(A)+ RNA the transport activity was identified in the 3.0- to 5.0-kb fractions, which were used for construction of a cDNA library. The library was screened for expression of cefadroxil transport after injection of complementary RNA synthesized in vitro from different pools of clones. A single clone (rPepT2) was isolated that stimulated cefadroxil uptake into oocytes approximately 70-fold at a pH of 6.0. Kinetic analysis of cefadroxil uptake expressed by the transporter's complementary RNA showed a single saturable high-affinity transport system shared by dipeptides, tripeptides, and selected amino-beta-lactam antibiotics. Electrophysiological studies established that the transport activity is electrogenic and affected by membrane potential. Sequencing of the cDNA predicts a protein of 729 amino acids with 12 membrane-spanning domains. Although there is a significant amino acid sequence identity (47%) to the recently cloned peptide transporters from rabbit and human small intestine, the renal transporter shows distinct structural and functional differences.

Amino Acid Sequence↗
PubMed1996

Electrogenic cation transport across leech caecal epithelium.

Electrogenic cation transport across the caecal epithelium of the leech Hirudo medicinalis was investigated using modified Ussing chambers. Transepithelial resistance (RT) and potential difference (VT) were 61.0 +/- 3.5 omega.cm2 and -1.1 +/- 0.2 mV (n = 149), respectively, indicating that leech caecal epithelium is a "leaky" epithelium. Under control conditions short circuit current (ISC) and transepithelial Na+ transport rate (INa) averaged at 22.1 +/- 1.5 microA.cm-2 and 49.7 +/- 2.6 microA.cm-2, respectively. Mucosal application of amiloride (100 mumol.l-1) or benzamil (50 mumol.l-1) influenced neither ISC nor INa. The transport system in the apical membrane showed no pronounced cation selectivity and a linear dependence on mucosal Na+ concentration. Removal of mucosal Ca2+ increased ISC by about 50% due to an increase of transepithelial Na+ transport. Trivalent cations (La3+ and Tb3+, 1 mmol.l-1 both) added to the mucosal Ringer solution reduced INa by more than 40%. Serosal ouabain (1 mmol.l-1) almost halved ISC and INa while 0.1% (= 5.4 mmol.l-1) DNP decreased INa to 11.8 +/- 5.1% of initial values. Serosal addition of cAMP increased both ISC and INa whereas the neurotransmitters. FMRFamide, acetylcholine, GABA, L-dopa, serotonin and dopamine failed to show any effects; octopamine, glycine and L-glutamate reduced INa markedly. On the basis of these results we conclude that in leech caecal epithelium apical uptake of monovalent cations is mediated by non-selective cation conductances which are sensitive to extracellular Ca2+ but insensitive to amiloride. Basolaterally Na+ is extruded via ouabain-sensitive and -insensitive ATPases. cAMP activates Na+ transport across leech caecal epithelium, although the physiological stimulus for cAMP-production remains unknown.

Animals↗
PubMed1996

Transport characteristics of differently charged cephalosporin antibiotics in oocytes expressing the cloned intestinal peptide transporter PepT1 and in human intestinal Caco-2 cells.

To investigate whether multiple peptide transporters mediate absorption of beta-lactams carrying different charges at physiological pH, we used the human intestinal cell line Caco-2 and Xenopus laevis oocytes expressing the cloned rabbit intestinal peptide transporter PepT1. Characteristics of transport of the anionic cefixime and the zwitterionic cefadroxil were assessed by 1) flux studies using radiolabeled compounds, by 2) measuring changes in pHin in cells and oocytes as a consequence of substrate-mediated proton influx and 3) by applying the two-electrode voltage clamp technique to assess the electrophysiological phenomena associated with beta-lactam transport in oocytes expressing PepT1. Both beta-lactams were rapidly taken up into Caco-2 cells and oocytes expressing PepT1 by a pH-dependent and saturable transport pathway. Mutual inhibition suggested that acidic and zwitterionic compounds may share a common transporter. Cefixime and cefadroxil caused a significant decline in intracellular pH as a consequence of proton coupled substrate influx. Uptake of cefixime and cefadroxil via PepT1 expressed in oocytes was electrogenic indicating that transport of both beta-lactams is associated with movement of net positive charge. The more acidic pH required for rheogenic cefixime uptake in both cell systems, when compared to cefadroxil uptake in both cell systems, when compared to cefadroxil uptake, and the concomitant faster intracellular acidification indicates that cefixime most likely is taken up only in its nonionized form with an additional proton being cotransported. This is supported by the observation that cefixime uptake at different pH correlated significantly with the percentage of the nonionized species being present. From our studies we conclude that a single peptide transport system can mediate electrogenic uptake of the neutral form of beta-lactam antibiotics into intestinal epithelial cells.

Animals↗
PubMed1996

[Benzamil and mucoviscidosis. Primary culture of nasal mucosa as an electrophysiologic in vitro model].

The genetic disease cystic fibrosis (CF) is characterized by a defect in the gene encoding for an epithelial chloride channel. This gene product, cystic fibrosis transmembrane conductance regulator (CFTR), in turn leads to a decreased chloride secretion. When this occurs sodium absorption is increased drastically because of an up-regulation of the epithelial sodium channel. In airway epithelia these changes in ion permeabilities result in dehydration of the mucus blanket and impaired mucociliary clearance, and subsequently lead to chronic infections of the airways. In this study we established a primary cell culture of human nasal epithelium from CF and non-CF patients. The increased transepithelial potential in CF exclusively was due to the greater sodium absorption. In previous studies, amiloride was found to specifically block the epithelial sodium channel. Inhalation of nebulized amiloride has been used clinically in the treatment of CF to inhibit sodium absorption and exert a secondary effect on water withdrawal. In the present cell culture model benzamil, an amiloride analogue, was found to inhibit sodium absorption completely, but at strikingly lower concentrations than amiloride. This raises the possibility of using benzamil for inhalation and to provide better efficacy in the symptomatic therapy of patients with CF.

Amiloride↗
PubMed1995

Amiloride-sensitive Na+ conductance in native Xenopus oocytes.

Endogenous Na+ conductances in the plasma membrane of oocytes of the South African clawed toad Xenopus laevis were investigated by microelectrode techniques and influx measurements. Removal of Na+ from the bath solution under voltage clamp conditions led to a decrease in the clamp current indicating the existence of native Na+ conductances. The observed current was voltage dependent but showed no marked rectification. Amiloride (10 microM) blocked this Na+ current reversibly. However, amiloride analogues such as benzamil and phenamil had no effect on this Na+ conductance. The Na+/H(+)-exchanger blocker EIPA (5-(N-ethyl-N-isopropyl)amiloride), another amiloride analogue, also had no effect thereby excluding a possible involvement of the Na+/H+ exchanger. The Na+ mediated current had a reversal potential of about 50 mV suggesting high selectivity of these Na+ conductances for Na+ over other monovalent cations. When Na+ was replaced by K+ in the bath solution, amiloride had no effect on the clamp current over the whole potential range demonstrating that only Na+ but not K+ can enter the cell via the investigated conductances. In radio tracer experiments 22Na+ influx into oocytes was nearly halved in presence of amiloride (10 microM), whereas benzamil and phenamil again failed to influence 22Na+ influx. These results suggest that the endogenous amiloride-sensitive Na+ conductance belongs to a new class of channels which is quite different from amiloride-sensitive epithelial Na+ channels.

Amiloride↗
PubMed1995

The Ca(2+)-induced leak current in Xenopus oocytes is indeed mediated through a Cl- channel.

Defolliculated oocytes of Xenopus laevis responded to removal of external divalent cations with large depolarizations and, when voltage clamped, with huge currents. Single channel analysis revealed a Cl- channel with a slope conductance of about 90 pS at positive membrane potentials with at least four substates. Single channel amplitudes and mean channel currents had a reversal potential of approximately -15 mV as predicted by the Nernst equation for a channel perfectly selective for Cl-. Readdition of Ca2+ immediately inactivated the channel and restored the former membrane potential or clamp current. The inward currents were mediated by a Ca2+ inactivated Cl- channel (CaIC). The inhibitory potency of Ca2+ was a function of the external Ca2+ concentration with a half maximal blocker concentration of about 20 microM. These channels were inhibited by the Cl- channel blockers flufenamic acid, niflumic acid and diphenylamine-2-carboxylate (DPC). In contrast, 4,4'-acetamido-4'-isothiocyanatostilbene-2, 2'-disulfonicacid (SITS), another Cl- channel blocker, led to activation of this Cl- channel. Like other Cl- channels, the CaIC was activated by cytosolic cAMP. Extracellular ATP inhibited the channel while ADP was without any effect. Injection of phorbol 12-myristate 13-acetate (PMA), a protein kinase C activating phorbol ester, stimulated the Cl- current. Cytochalasin D, an actin filament disrupting compound, reversibly decreased the clamp current demonstrating an influence of the cytoskeleton. The results indicate that removal of divalent cations activates Cl- channels in Xenopus oocytes which share several features with Cl- channels of the CLC family. The former so-called leak current of oocytes under divalent cation-free conditions is nothing else than an activation of Cl- channels.

1-Methyl-3-isobutylxanthine↗
PubMed1995

Influence of extracellular Ca2+ on endogenous Cl- channels in Xenopus oocytes.

Removal of Ca2+ from the external bath solution evoked marked depolarization and large currents (up to several microamperes) in voltage-clamped defolliculated oocytes of Xenopus laevis. The resulting current was not carried by a cation influx but was due to a huge Cl- efflux, which could be strongly inhibited by the Cl- channel blockers flufenamic acid and niflumic acid. Removal of Mg2+ or Ba2+ from the solutions had the same effects as removing Ca2+. The reversal potential of -12 mV also indicated that Cl- channels were responsible for the large currents. Patch-clamp studies revealed a single-channel slope conductance of 90 pS. During oocyte maturation these channels remained active. The half-maximal Ca2+ concentration of about 20 microM showed that quite low doses of extracellular Ca2+ profoundly influence the electrical properties of the oocyte membrane.

Animals↗
PubMed1995

Regulation of electrogenic Na+ transport across leech skin.

The dorsal integument of the medical leech Hirudo medicinalis exhibits a marked amiloride-sensitive Na+ absorption. With 20 mM Na+ in the apical solution, the transepithelial short-circuit current (Isc) was approximately 40% higher than with 115 mM Na+, whereas the transepithelial potential (VT) with 20 mM Na+ was -35.7 +/- 4.5 and -20.6 +/- 2.6 mV with 115 mM Na+. Amiloride (100 microM) inhibition at 20 mM apical Na+ was also significantly larger than with 115 mM Na+ in the solution. Benzamil (100 microM) showed additional inhibition after amiloride. Large transient overshooting currents occurred only when 115 mM Na+ was added after some minutes of Na(+)-free apical solution. Addition of adenosine 3',5'-cyclic monophosphate (cAMP) to the serosal side in the presence of 115 mM apical Na+ nearly doubled Isc. This cAMP effect was reduced to only 20% in the presence of 20 mM Na+. Guanosine 3',5'-cyclic monophosphate (cGMP) slightly increased Isc, whereas ATP showed biphasic potency. Removal of calcium from the apical side resulted in a large stimulation of amiloride-sensitive Isc only in the presence of 115 mM Na+. When currents were activated with cAMP, a deprivation of Ca2+ modestly reduced the amiloride-sensitive Isc. The Na+ channel of leech integument was found highly selective for Na+ over other monovalent cations. The permeability ratio for Na+ over K+ was approximately 30:1; the selectivity relationship for the investigated cations was Na+ > Li+ > NH4+ > K+ approximately Cs+ approximately Rb+.

Amiloride↗
PubMed1994

Expression cloning of a cDNA from rabbit small intestine related to proton-coupled transport of peptides, beta-lactam antibiotics and ACE-inhibitors.

Injection of poly(A)+ RNA from rabbit small intestine into Xenopus laevis oocytes resulted in expression of pH dependent transport of the aminocephalosporin cefadroxil. A cDNA library constructed from a 2.2 to 5 kb fraction was screened for expression of cefadroxil transport after injection of the corresponding cRNA synthetized in vitro from different pools of clones. The single clone identified stimulated uptake of cefadroxil into oocytes about 50-fold at pH 6.5. Kinetic analysis of expressed transport activity revealed a saturable transport system shared by amino beta-lactam antibiotics, dipeptides and selected angiotensin converting enzyme inhibitors. Evidence for rheogenic cefadroxil/H(+)-cotransport was obtained by a) The demonstration that cefadroxil influx increased the inward current in oocytes clamped at a holding potential of -60 mV in sodium-free medium and b) A decrease of intracellular pH in oocytes caused by cefadroxil uptake. Current-voltage relationships in the presence of glycylsarcosine or cefadroxil showed that transport activity is dependent on the membrane potential. Sequencing of the cDNA revealed its identity with the recently cloned peptide transporter from rabbit small intestine designated PepT1.

Angiotensin-Converting Enzyme Inhibitors↗
PubMed1993

Ion transport across leech integument. I. Electrogenic Na+ transport and current fluctuation analysis of the apical Na+ channel.

The dorsal skin of the leech Hirudo medicinalis was used for electrophysiological measurements performed in Ussing chambers. The leech skin is a tight epithelium (transepithelial resistance = 10.5 +/- 0.5 k omega.cm-2) with an initial short-circuit current of 29.0 +/- 2.9 microA.cm-2. Removal of Na+ from the apical bath medium reduced short-circuit current about 55%. Ouabain (50 mumol.l-1) added to the basolateral solution, depressed the short-circuit current completely. The Na+ current saturated at a concentration of 90 mmol Na+.l-1 in the apical solution (KM = 11.2 +/- 1.8 mmol.l-1). Amiloride (100 mumol.l-1) on the apical side inhibited ca. 40% of the Na+ current and indicated the presence of Na+ channels. The dependence of Na+ current on the amiloride concentration followed Michaelis-Menten kinetics (Ki = 2.9 +/- 0.4 mumol.l-1). The amiloride analogue benzamil had a higher affinity to the Na+ channel (Ki = 0.7 +/- 0.2 mumol.l-1). Thus, Na+ channels in leech integument are less sensitive to amiloride than channels known from vertebrate epithelia. With 20 mmol Na+.l-1 in the mucosal solution the tissue showed an optimum amiloride-inhibitable current, and the amiloride-sensitive current under this condition was 86.8 +/- 2.3% of total short-circuit current. Higher Na+ concentrations lead to a decrease in amiloride-blockade short-circuit current. Stimulation of the tissue with cyclic adenosine monophosphate (100 mumol.l-1) and isobutylmethylxanthine (1 mmol.l-1) nearly doubled short-circuit current and increased amiloride-sensitive Na+ currents by 50%.(ABSTRACT TRUNCATED AT 250 WORDS)

Amiloride↗
PubMed1992

Expression of amiloride-sensitive Na+ channels of hen lower intestine in Xenopus oocytes: electrophysiological studies on the dependence of varying NaCl intake.

Epithelial Na+ channels were incorporated into the plasma membrane of Xenopus laevis oocytes after micro-injection of RNA from hen lower intestinal epithelium (colon and coprodeum). The animals were fed either a normal poultry food which contained NaCl (HS), or a similar food devoid of NaCl (LS). Oocytes were monitored for the expression of amiloride-sensitive sodium channels by measuring membrane potentials and currents. Oocytes injected with poly(A)+RNA prepared from HS animals or non-injected control oocytes showed no detectable sodium currents, whereas oocytes injected with LS-poly(A)+RNA had large amiloride-blockable sodium currents. These currents were almost completely saturated by sodium concentrations of 20 mM with a Km of about 2.6 mM sodium. Amiloride (10 microM) inhibits the expressed sodium channels entirely and examination of dose response relationships yielded a half-maximal inhibition concentration (Ki) of 120 nM amiloride. I-V difference curves in the presence or absence of sodium or amiloride (10 microM) indicate a potential dependence of the sodium transport which can be described by the Goldman equation. When Na+ is replaced by K+, no amiloride response was detected indicating a high selectivity for Na+ over K+. These results provide strong evidence that intestinal Na+ channels are regulated by dietary salt intake on the RNA level.

Amiloride↗
PubMed1991

Comparison of a Na+/D-glucose cotransporter from rat intestine expressed in oocytes of Xenopus laevis with the endogenous cotransporter.

Epithelial Na+/D-glucose cotransport was incorporated into the plasma membrane of Xenopus oocytes after microinjection of poly(A)(+)-mRNA from rat intestine tissue and was detected by measurements of uptake of [14C]AMG (methyl alpha-D-glucopyranoside). In mRNA-injected oocytes, the rate of AMG uptake exceeds the rate of endogenous Na+/AMG cotransport by a factor of up to 30. It is demonstrated that the additionally expressed transport differs qualitatively from the endogenous transport with respect to several parameters which is a prerequisite for the demonstration of expression of a foreign transporter: (1) The expressed system is more sensitive to external glucose or AMG and to the specific inhibitor phlorizin, (2) it is less sensitive to external Na+ and to changes in membrane potential, and (3) it is susceptible to inhibition by monoclonal antibodies, known to bind specifically to Na+/glucose cotransporters and to modulate the cotransport in kidney and intestine. The use of the antibodies allows one to distinguish between endogenous Na+/AMG cotransport and foreign cotransport expressed by injection of foreign mRNA. The expression of the foreign transport leads to transport rates that are high enough to detect the electrical current generated by the Na+/glucose cotransport. This allows future characterization of the cotransport system under voltage-clamp conditions by analyzing membrane current.

Animals↗
PubMed1989

Endogenous D-glucose transport in oocytes of Xenopus laevis.

Endogenous glucose uptake by the oocytes of Xenopus laevis consists of two distinct components: one that is independent of extracellular Na+, and the other one that represents Na+-glucose cotransport. The latter shows similar characteristics as 2 Na+-1 glucose cotransport of epithelial cells: The similarities include the dependencies on external concentrations of Na+, glucose, and phlorizin, and on pH. As in epithelial cells, the glucose uptake in oocytes can also be stimulated by lanthanides. Both the electrogenic cotransport and the inhibition by phlorizin are voltage-dependent; the data are compatible with the assumption that the membrane potential acts as a driving force for the reaction cycle of the transport process. In particular, hyperpolarization seems to stimulate transport by recruitment of substrate binding sites to the outer membrane surface. The results described pertain to oocytes arrested in the prophase of the first meiotic division; maturation of the oocytes leads to a downregulation of both the Na+-independent and the Na+-dependent transport systems. The effect on the Na+-dependent cotransport is the consequence of a change of driving force due to membrane depolarization associated with the maturation process.

Animals↗
PubMed1988

What right to die?

This letter first discusses two meanings of a "right to die." In the popular sense, the term refers to a right to refuse life-sustaining treatment. In the strict sense, the term signifies an affirmative right to obtain death--a right to suicide. The letter then explores the legal implications of a suicide right. This right would extend to competent adults, mature minors, and probably also incompetent persons. Counselors would have to inform clients of the suicide option. Intervention to prevent suicide could trigger civil liability. Suicidal intentions would not justify involuntary commitment. Consent would become a defense to homicide.

Humans↗