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Biomedical subjects

F Hofmann

Publications and source records attributed to F Hofmann.

At least 109 records · Page 6Linked to original sources
PubMed1999

Reconstruction of the dihydropyridine site in a non-L-type calcium channel: the role of the IS6 segment.

Mutations of eight to nine amino acids of IIIS5, IIIS6 and IVS6 segments were shown to reconstruct the dihydropyridine (DHP) interaction site in the non-L-type alpha1E or alpha1A calcium channels. The reconstructed site enabled enantiomer-selective inhibition and activation of the expressed chimeras by DHPs but failed to transfer voltage dependence of the current inhibition. Here we show that transfer of four non-conserved amino acids from the IS6 segment to the DHP-sensitive alpha1E chimera increased the inhibition by (+)isradipine at the hyperpolarized membrane potential of -100 mV and enhanced the voltage-dependent block.

Animals↗
PubMed1999

Electric field pulses can induce apoptosis.

Injection of electric field pulses of high intensity (kV/cm) and short duration (microsecond range) into a cell suspension results in a temporary increase of the membrane permeability due to a reversible electric breakdown of the cell membrane. Here we demonstrate that application of supercritical field pulses between 4. 5 and 8.1 kV/cm strength and 40 microsec duration induce typical features of apoptosis in Jurkat T-lymphoblasts and in HL-60 cells including DNA fragmentation and cleavage of the poly(ADP ribose) polymerase. Apoptosis induction did not depend on the presence of any particular electrolyte in the extracellular medium. However, no apoptosis was observed in solutions without a minimum amount of salt. Apoptotic DNA fragmentation was prevented by the caspase inhibitor zVAD.

Apoptosis↗
PubMed1999

Two pacemaker channels from human heart with profoundly different activation kinetics.

Cardiac pacemaking is produced by the slow diastolic depolarization phase of the action potential. The hyperpolarization-activated cation current (If) forms an important part of the pacemaker depolarization and consists of two kinetic components (fast and slow). Recently, three full-length cDNAs encoding hyperpolarization-activated and cyclic nucleotide-gated cation channels (HCN1-3) have been cloned from mouse brain. To elucidate the molecular identity of cardiac pacemaker channels, we screened a human heart cDNA library using a highly conserved neuronal HCN channel segment and identified two cDNAs encoding HCN channels. The hHCN2 cDNA codes for a protein of 889 amino acids. The HCN2 gene is localized on human chromosome 19p13.3 and contains eight exons spanning approximately 27 kb. The second cDNA, designated hHCN4, codes for a protein of 1203 amino acids. Northern blot and PCR analyses showed that both hHCN2 and hHCN4 are expressed in heart ventricle and atrium. When expressed in HEK 293 cells, either cDNA gives rise to hyperpolarization-activated cation currents with the hallmark features of native If. hHCN2 and hHCN4 currents differ profoundly from each other in their activation kinetics, being fast and slow, respectively. We thus conclude that hHCN2 and hHCN4 may underlie the fast and slow component of cardiac If, respectively.

Biological Clocks↗
PubMed1999

Absence of modulation of the expressed calcium channel alpha1G subunit by alpha2delta subunits.

1. The modulatory action of the alpha2delta subunit on various high-voltage-activated calcium channels has been demonstrated previously. However, very little is known about auxiliary subunit modulation of low-voltage-activated (LVA) calcium channels. We have examined the modulation of the alpha1G subunit corresponding to the neuronal T-type calcium channel by the ubiquitously expressed alpha2delta-1 and brain-specific alpha2delta-3 subunits. 2. The alpha1G subunit was expressed alone or in combination with either the alpha2delta-1 or alpha2delta-3 subunit in human embryonic kidney (HEK 293) cells and whole-cell barium currents were measured. The current density-voltage relationships for peak and sustained current, kinetics of current activation and inactivation, voltage dependence of current inactivation and time course of the recovery from inactivation were analysed for each type of expressed channel. No significant difference was found for any of the examined parameters. 3. These results suggest that the LVA alpha1G channel is not regulated by known auxiliary alpha2delta subunits.

Animals↗
PubMed1999

Increased adhesion and aggregation of platelets lacking cyclic guanosine 3',5'-monophosphate kinase I.

Atherosclerotic vascular lesions are considered to be a major cause of ischemic diseases, including myocardial infarction and stroke. Platelet adhesion and aggregation during ischemia-reperfusion are thought to be the initial steps leading to remodeling and reocclusion of the postischemic vasculature. Nitric oxide (NO) inhibits platelet aggregation and smooth muscle proliferation. A major downstream target of NO is cyclic guanosine 3', 5'-monophosphate kinase I (cGKI). To test the intravascular significance of the NO/cGKI signaling pathway in vivo, we have studied platelet-endothelial cell and platelet-platelet interactions during ischemia/reperfusion using cGKI-deficient (cGKI-/-) mice. Platelet cGKI but not endothelial or smooth muscle cGKI is essential to prevent intravascular adhesion and aggregation of platelets after ischemia. The defect in platelet cGKI is not compensated by the cAMP/cAMP kinase pathway supporting the essential role of cGKI in prevention of ischemia-induced platelet adhesion and aggregation.

Animals↗
PubMed1999

Molecular diversity of the calcium channel alpha2delta subunit.

Sequence database searches with the alpha2delta subunit as probe led to the identification of two new genes encoding proteins with the essential properties of this calcium channel subunit. Primary structure comparisons revealed that the novel alpha2delta-2 and alpha2delta-3 subunits share 55.6 and 30.3% identity with the alpha2delta-1 subunit, respectively. The number of putative glycosylation sites and cysteine residues, hydropathicity profiles, and electrophysiological character of the alpha2delta-3 subunit indicates that these proteins are functional calcium channel subunits. Coexpression of alpha2delta-3 with alpha1C and cardiac beta2a or alpha1E and beta3 subunits shifted the voltage dependence of channel activation and inactivation in a hyperpolarizing direction and accelerated the kinetics of current inactivation. The kinetics of current activation were altered only when alpha2delta-1 or alpha2delta-3 was expressed with alpha1C. The effects of alpha2delta-3 on alpha1C but not alpha1E are indistinguishable from the effects of alpha2delta-1. Using Northern blot analysis, it was shown that alpha2delta-3 is expressed exclusively in brain, whereas alpha2delta-2 is found in several tissues. In situ hybridization of mouse brain sections showed mRNA expression of alpha2delta-1 and alpha2delta-3 in the hippocampus, cerebellum, and cortex, with alpha2delta-1 strongly detected in the olfactory bulb and alpha2delta-3 in the caudate putamen.

Amino Acid Sequence↗
PubMed1999

The role of subunit composition on prepulse facilitation of the cardiac L-type calcium channel.

Facilitation of calcium current by depolarizing prepulses has been observed in many cells including cardiac muscle. The mechanism underlying prepulse facilitation is controversial with respect to the requirements of channel subunits and cAMP kinase. We found that coexpression of the cardiac alpha1C-a subunit with the cardiac beta2a subunit significantly promotes the facilitation of I(Ba) by strong depolarizing prepulses. The magnitude of I(Ba) facilitation depended on the voltage potential of the prepulse and the interval duration between prepulse and test pulse. Prepulse facilitation was not affected by coexpression of AKAP79 and conditions favoring cAMP-dependent phosphorylation. Prepulse facilitation was also observed in cells expressing an alpha1C-a subunit which was truncated at residue 1733 removing the cAMP kinase site at Ser-1928. Facilitation was abolished by coexpression of the alpha2delta-1 or alpha2delta-3 subunit. We conclude that the expressed alpha1C-a beta2a complex is sufficient to support prepulse facilitation. Facilitation is prevented by coexpression of the alpha2delta subunit.

A Kinase Anchor Proteins↗
PubMed1999

Long-term potentiation in the hippocampal CA1 region of mice lacking cGMP-dependent kinases is normal and susceptible to inhibition of nitric oxide synthase.

Long-term potentiation (LTP) is a potential cellular mechanism for learning and memory. The retrograde messenger nitric oxide (NO) is thought to induce LTP in the CA1 region of the hippocampus via activation of soluble guanylyl cyclase (sGC) and, ultimately, cGMP-dependent protein kinase (cGK). Two genes code for the isozymes cGKI and cGKII in vertebrates. The functional role of cGKs in LTP was analyzed using mice lacking the gene(s) for cGKI, cGKII, or both. LTP was not altered in the mutant mice lineages. However, LTP was reduced by inhibition of NO synthase and NMDA receptor antagonists, respectively. The reduced LTP was not recovered by the cGK-activator 8-(4 chlorophenylthio)-cGMP. Moreover, LTP was not affected by the sGC inhibitor 1H-[1,2,4]oxadiazolo[4,3-a]-quiloxalin-1-one. In contrast, it was effectively suppressed by nicotinamide, a blocker of the ADP-ribosyltransferase. These results show that cGKs are not involved in LTP in mice and that NO induces LTP through an alternative cGMP-independent pathway, possibly ADP-ribosylation.

Animals↗
PubMed1999

Interaction of three structurally distinct Ca2+ channel activators with single L-type Ca2+ channels.

The dihydropyridine S(-)-Bay K 8644 (Bay K), the benzoylpyrrole FPL 64176 (FPL) and the benzodiazocine CGP 48506 (CGP) are structurally unrelated L-type Ca2+ channels agonists. The aim of our study was to investigate whether these three drugs interact with different binding sites and thereby modulate the behaviour of L-type Ca2+ channels in a qualitatively different manner. Single-channel recordings were performed on CHO cells stably expressing the alpha1C-b subunit of the L-type Ca2+ channel. Mean open time and open probability were determined sweep by sweep and the effects of CGP (10(-4) M), Bay K (10(-6) M) and FPL (10(-6) M) were compared. All three compounds increased mean open time and open probability when applied alone. However, the gating pattern changes induced by each drug were qualitatively and quantitatively different. We also applied binary mixtures and analysed the resulting sweeps with respect to their gating pattern. The application of mixtures did result in a gating pattern not seen with any of the single drugs. The mixture of CGP and FPL led to a prolonged mean open time compared with each single drug. The mixture of Bay K and FPL exhibited an open probability lower than with each single drug. The mixture of CGP and Bay K increased the mean open time per sweep like Bay K, but the number of openings was similar to the level seen with CGP alone. These results cannot be explained by assuming alternative binding of the drugs to a single binding site. We therefore conclude that Bay K, CGP and FPL bind to different but interacting sites on the L-type Ca2+ channel.

3-Pyridinecarboxylic acid, 1,4-dihydro-2,6-dimethy↗
PubMed1999

A T-type calcium channel from mouse brain.

A member of the low-voltage-activated calcium channel family was identified in mouse brain by taking advantage of amino acid sequences that have been evolutionary conserved. The identified sequence is similar to that of the recently cloned rat alpha1G T-type calcium channel, but there are differences in two insertions in the intracellular connecting loops. Northern blot analysis indicates that its expression is strong in the brain. In situ hybridization revealed that, in mouse brain, the alpha1G mRNA is found in the cerebellum, hippocampus, thalamus and olfactory bulb. In contrast to L-type calcium channel currents, IBa and ICa through the alpha1G channel expressed in HEK293 cells did not differ in terms of current density, voltage dependence of current activation, inactivation and deactivation, and speed of recovery from voltage-dependent inactivation. The kinetics of ICa inactivation were significantly slower than those of IBa. The expressed alpha1G channel has a relatively high sensitivity to mibefradil, but is only slightly affected by Ni2+.

Amino Acid Sequence↗
PubMed1999

Dramatic inhibition of retinal and choroidal neovascularization by oral administration of a kinase inhibitor.

The most common cause of new blindness in young patients is retinal neovascularization, and in the elderly is choroidal neovascularization. Therefore, there has been a great deal of attention focused on the development of new treatments for these disease processes. Previous studies have demonstrated partial inhibition of retinal neovascularization in animal models using antagonists of vascular endothelial growth factor or other signaling molecules implicated in the angiogenesis cascade. These studies have indicated potential for drug treatment, but have left many questions unanswered. Is it possible to completely inhibit retinal neovascularization using drug treatment with a mode of administration that is feasible to use in patients? Do agents that inhibit retinal neovascularization have any effect on choroidal neovascularization? In this study, we demonstrate complete inhibition of retinal neovascularization in mice with oxygen-induced ischemic retinopathy by oral administration of a partially selective kinase inhibitor that blocks several members of the protein kinase C family, along with vascular endothelial growth factor and platelet-derived growth factor receptor tyrosine kinases. The drug also blocks normal vascularization of the retina during development but has no identifiable adverse effects on mature retinal vessels. In addition, the kinase inhibitor causes dramatic inhibition of choroidal neovascularization in a laser-induced murine model. These data provide proof of concept that pharmacological treatment is a viable approach for therapy of both retinal and choroidal neovascularization.

Administration, Oral↗
PubMed1999

Computer-assisted animation of atrial tachyarrhythmias recorded with a 64-electrode basket catheter.

OBJECTIVES: The aim of this study was to assess the value of a new mapping technique based on computer-assisted animation of multielectrode basket catheter (BC) recordings in patients with atrial arrhythmias. BACKGROUND: The three-dimensional activation patterns of cardiac arrhythmias are not completely understood owing to limitations of conventional mapping techniques. METHODS: The study included 32 patients with atrial tachycardia (AT) and 38 patients with atrial flutter (AFL). A software program was developed to analyze the activation patterns based on 56 bipolar electrograms recorded with a 64-electrode BC deployed in the right atrium (RA). RESULTS: The total time needed for the animation of activation patterns of atrial arrhythmias was 5 +/- 0.8 min. In 22 patients with right AT, the animated maps revealed that arrhythmia was unifocal in 15 patients, multifocal in 2 patients, polymorphic in 4 patients and reentrant in 1 patient. In 10 patients with left AT, breakthroughs on the right side of the septum (2 in 8 patients and 1 in 2 patients) and a left-to-right activation of the RA were demonstrated. In patients with typical AF, the reentrant excitation was a broad activation front with preferential propagation around the tricuspid annulus. In patients with atypical AFL, the reentry circuit involved one of the venae cavae and a line of block located in the posterior wall. CONCLUSIONS: The computer-assisted animation of multiple electrograms recorded with a BC is a valuable mapping tool that delineates the three-dimensional activation patterns of various atrial arrhythmias. The technique is appropriate for complex, short-lived or unstable arrhythmias.

Adult↗
PubMed1999

Rundown of somatodendritic N-methyl-D-aspartate (NMDA) receptor channels in rat hippocampal neurones: evidence for a role of the small GTPase RhoA.

Actin filament (F-actin) depolymerization leads to the use-dependent rundown of N-methyl-D-aspartate (NMDA) receptor activity in rat hippocampal neurones. Depolymerization is promoted by Ca2+ which enters the cells via NMDA receptor channels. The ras homologue (Rho) GTPases (RhoA, Rac1 and Cdc42) promote actin polymerization and thus control the actin cytoskeleton. We have investigated, by means of the whole-cell patch clamp technique, whether the actin fibres which interact with NMDA receptors are controlled by Rho GTPases. In the presence of intracellular ATP which attenuates rundown, the C3 toxin from Clostridium (C.) botulinum was used to inactivate RhoA. Indeed, it enhanced the use-dependent rundown of NMDA-evoked inward currents to a level similar to that obtained in the absence of ATP. Lethal toxin from Clostridium sordellii which inactivates Rac1 and Cdc42 lacked this effect. We suggest that the function of somatodendritic NMDA receptor channels in rat hippocampal neurones can be modulated by RhoA via its action on F-actin.

Actins↗