[Instructions after vaccination].
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Humans↗
Biomedical subjects
F Hofmann
Publications and source records attributed to F Hofmann.
Dihydropyridine enantiomers block recombinant L-type Ca2+ channels by two different mechanisms.
1. The molecular basis of the state-dependent block of L-type Ca2+ channels by dihydropyridines is still poorly understood. Therefore, we studied the enantioselectivity of Ca2+ channel block by isradipine enantiomers at three holding potentials (-80, -60 and -40 mV) in Chinese hamster ovary (CHO) cells stably expressing the rabbit lung alpha1C-b-subunit. 2. The extent of enantioselectivity did not markedly change with the holding potential (IC50 ratios of 104-138), whereas the potency of both isradipine enantiomers increased with depolarisation of the holding potential. 3. In addition to its block of the peak Ca2+ channel current, Ipeak, (-)-isradipine inhibited the relative current at the end of the test pulse, the so-called Ilate, normalised to Ipeak (Ilate/Ipeak). This effect was unaffected by the holding potential and revealed distinct kinetics compared to the development of conventional block of Ipeak. 4. When these effects were studied using an alpha1C-b-mutant lacking the high-affinity dihydropyridine binding site, expressed in human embryonic kidney (HEK 293) cells, both enantiomers blocked Ilate/Ipeak to a similar degree. 5. Our data are discussed within the framework of the 'guarded receptor' and the 'modulated receptor' hypotheses. The very different properties of the block of Ilate/Ipeak compared to those of the conventional high-affinity block of Ipeak suggest the existence of an additional mechanism possibly mediated via a second, distinct binding site.
Animals↗
Mechanisms and kinetics of procathepsin D activation.
In vitro, procathepsin D is activated to pseudocathepsin D by incubation at low pH. To investigate the mechanism of this activation, recombinant human procathepsin D and two mutants were generated in a baculovirus expression system. One mutant carried a point mutation within the catalytic domain, which resulted in a catalytically inactive enzyme form (D77A). The other carried a point mutation within the propeptide, which prevented activation by processing at the 'autoproteolysis-site' (L26P). Neither mutant is capable of processing itself to form pseudocathepsin D, and L26P is not able to process D77A. Despite the inability of L26P to cleave either its own or a wild-type prosequence, it did exhibit activity against a synthetic peptide substrate. The ability of intact precursor (zymogen) to cleave a peptide, but not a protein substrate, offers new insights into the mechanism of inhibition by the propeptide. Mature cathepsin D can process the inactive D77A mutant to the pseudoform, demonstrating that processed species are capable of cleaving zymogen molecules in an intermolecular interaction. In addition, kinetic studies provide evidence for a two-phase mechanism for the conversion of procathepsin D to pseudocathepsin D, one phase where the first molecules of pseudocathepsin D are formed at a low rate and a second phase where the process is autocatalytically accelerated by newly formed pseudocathepsin D molecules. Finally, with the help of the mutants L26P and D77A it was observed that at least two additional proteinase activities, found in conditioned media from insect cell culture, are capable of activating procathepsin D by cleaving it within the proregion. This observation suggests that there are likely to be multiple proteinases in the extracellular matrix that are capable of activating procathepsin D, thereby triggering the second autocatalytic phase. This may also be important for solid tumors, where the presence of cathepsin D has been correlated with tumor growth and invasion.
Amino Acid Sequence↗
Selective loss of cone function in mice lacking the cyclic nucleotide-gated channel CNG3.
Two types of photoreceptors, rods and cones, coexist in the vertebrate retina. An in-depth analysis of the retinal circuitry that transmits rod and cone signals has been hampered by the presence of intimate physical and functional connections between rod and cone pathways. By deleting the cyclic nucleotide-gated channel CNG3 we have generated a mouse lacking any cone-mediated photoresponse. In contrast, the rod pathway is completely intact in CNG3-deficient mice. The functional loss of cone function correlates with a progressive degeneration of cone photoreceptors but not of other retinal cell types. CNG3-deficient mice provide an animal model to dissect unequivocally the contribution of rod and cone pathways for normal retinal function.
Animals↗
Inhibition of amylase secretion from differentiated AR4-2J pancreatic acinar cells by an actin cytoskeleton controlled protein tyrosine phosphatase activity.
Disruption of the actin cytoskeleton in AR4-2J pancreatic acinar cells led to an increase in cytosolic protein tyrosine phosphatase activity, abolished bombesin-induced tyrosine phosphorylation and reduced bombesin-induced amylase secretion by about 45%. Furthermore, both tyrosine phosphorylation and amylase secretion induced by phorbol ester-induced activation of protein kinase C were abolished. An increase in the cytosolic free Ca2+ concentration by the Ca2+ ionophore A23187 had no effect on tyrosine phosphorylation but induced amylase release. Only when added together with phorbol ester, the same level of amylase secretion as with bombesin was reached. This amylase secretion was inhibited by about 40%, by actin cytoskeleton disruption similar to that induced by bombesin. We conclude that actin cytoskeleton-controlled protein tyrosine phosphatase activity downstream of protein kinase C activity regulates tyrosine phosphorylation which in part is involved in bombesin-stimulated amylase secretion.
Actins↗
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↗
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↗
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↗
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↗
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↗
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↗
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↗
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↗
Hyperpolarization-activated cation channels: a multi-gene family.
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Amino Acid Sequence↗
Voltage-dependent calcium channels: from structure to function.
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Amino Acid Sequence↗
Structure and function of cGMP-dependent protein kinases.
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Animals↗
Structure and function of cyclic nucleotide-gated channels.
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Animals↗
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↗