Biomedical subjects

Franz Hofmann

Publications and source records attributed to Franz Hofmann.

At least 73 records · Page 4Linked to original sources

PubMed2003

Direct activation of PDE5 by cGMP: long-term effects within NO/cGMP signaling.

In platelets, the nitric oxide (NO)-induced cGMP response is indicative of a highly regulated interplay of cGMP formation and cGMP degradation. Recently, we showed that within the NO-induced cGMP response in human platelets, activation and phosphorylation of phosphodiesterase type 5 (PDE5) occurred. Here, we identify cyclic GMP-dependent protein kinase I as the kinase responsible for the NO-induced PDE5 phosphorylation. However, we demonstrate that cGMP can directly activate PDE5 without phosphorylation in platelet cytosol, most likely via binding to the regulatory GAF domains. The reversal of activation was slow, and was not completed after 60 min. Phosphorylation enhanced the cGMP-induced activation, allowing it to occur at lower cGMP concentrations. Also, in intact platelets, a sustained NO-induced activation of PDE5 for as long as 60 min was detected. Finally, the long-term desensitization of the cGMP response induced by a low NO concentration reveals the physiological relevance of the PDE5 activation within NO/cGMP signaling. In sum, we suggest NO-induced activation and phosphorylation of PDE5 as the mechanism for a long-lasting negative feedback loop shaping the cGMP response in human platelets in order to adapt to the amount of NO available.

3',5'-Cyclic-GMP Phosphodiesterases↗

PubMed2003

Dominant-negative suppression of HCN channels markedly reduces the native pacemaker current I(f) and undermines spontaneous beating of neonatal cardiomyocytes.

BACKGROUND: The pacemaker current I(f) contributes to spontaneous diastolic depolarization of cardiac autonomic cells. In heterologous expression, HCN channels exhibit a hyperpolarization-activated inward current similar to I(f). However, the links between HCN genes and native I(f) are largely inferential, and it remains unknown whether I(f) is essential for cardiac pacing. METHODS AND RESULTS: To clarify this situation, we generated a GYG(402-404)AYA pore mutation of HCN2, which rendered the channel nonfunctional and suppressed wild-type HCN2 in a dominant-negative manner in Chinese hamster ovary cells. In addition, HCN2-AYA suppressed I(HCN4) in a dominant-negative manner when coexpressed with wild-type HCN4, indicating that the 2 isoforms HCN2 and HCN4 are able to coassemble to form heteromultimeric complexes. Given that HCN2 and HCN4 are the dominant HCN mRNA transcripts in neonatal rat ventricle, we expressed HCN2-AYA in neonatal cardiocytes using adenoviral gene transfer to test the effect of HCN suppression on native I(f). I(f) density was indeed reduced markedly, from 7.8+/-1.6 pA/pF (n=13) in control cells to 0.3+/-0.2 pA/pF (n=11) in HCN2-AYA-infected cells when measured at -130 mV (P<0.001). To probe the effect of HCN on cardiac pacing, we infected spontaneously beating neonatal monolayers with adenoviral vectors expressing wild-type and mutant HCN channels. Infection with HCN2 and HCN4 accelerated the beating rate significantly, to 230.5+/-8.6 bpm (n=12) and 223.5+/-12.3 bpm (n=10), respectively, compared with control cultures (83.4+/-4.5 bpm, n=13, P<0.001). Conversely, HCN2-AYA completely undermined spontaneous pacing of neonatal cardiocytes. CONCLUSIONS: HCN channels are the major molecular component of native I(f) and are critical for spontaneous beating of neonatal cardiomyocytes.

Adenoviridae↗

PubMed2003

Absence epilepsy and sinus dysrhythmia in mice lacking the pacemaker channel HCN2.

Hyperpolarization-activated cation (HCN) channels are believed to be involved in the generation of cardiac pacemaker depolarizations as well as in the control of neuronal excitability and plasticity. The contributions of the four individual HCN channel isoforms (HCN1-4) to these diverse functions are not known. Here we show that HCN2-deficient mice exhibit spontaneous absence seizures. The thalamocortical relay neurons of these mice displayed a near complete loss of the HCN current, resulting in a pronounced hyperpolarizing shift of the resting membrane potential, an altered response to depolarizing inputs and an increased susceptibility for oscillations. HCN2-null mice also displayed cardiac sinus dysrhythmia, a reduction of the sinoatrial HCN current and a shift of the maximum diastolic potential to hyperpolarized values. Mice with cardiomyocyte- specific deletion of HCN2 displayed the same dysrhythmia as mice lacking HCN2 globally, indicating that the dysrhythmia is indeed caused by sinoatrial dysfunction. Our results define the physiological role of the HCN2 subunit as a major determinant of membrane resting potential that is required for regular cardiac and neuronal rhythmicity.

Animals↗

PubMed2003

A stimulatory role for cGMP-dependent protein kinase in platelet activation.

It is currently accepted that cGMP-dependent protein kinase (PKG) inhibits platelet activation. Here, we show that PKG plays an important stimulatory role in platelet activation. Expression of recombinant PKG in a reconstituted cell model enhanced von Willebrand factor (vWF)-induced activation of the platelet integrin alpha(IIb)beta(3). PKG knockout mice showed impaired platelet responses to vWF or low doses of thrombin and prolonged bleeding time. Human platelet aggregation induced by vWF or low-dose thrombin was inhibited by PKG inhibitors but enhanced by cGMP. Furthermore, a cGMP-enhancing agent, sildenafil, promoted vWF- or thrombin-induced platelet aggregation. The cGMP-stimulated platelet responses are biphasic, consisting of an initial transient stimulatory response that promotes platelet aggregation and a subsequent inhibitory response that limits the size of thrombi.

Animals↗

PubMed2003

Association of phospholamban with a cGMP kinase signaling complex.

The cGMP kinase signaling complex identified previously in tracheal smooth muscle membranes contains a number of cGMP kinase substrates termed G0 through G4. G0, G1, and G2 were identified as IP(3) receptor I (IP(3)RI), IRAG, and cGMP kinase I. Sequencing of purified G3 and G4 showed that these proteins were proteolytic cleavage products of IRAG. However, the purified cGMP kinase signaling complex contained following additional proteins: alpha-actin, calponin H1, and phospholamban (PLB) as verified by MALDI-TOF as well as MS/MS sequencing and immune detection. The complex of these six proteins was immune precipitated by antibodies to each protein. The proteins were phosphorylated by the endogenous cGMP kinase I with the exception of alpha-actin and calponin H1. The complex did not contain the Ca(2+)-ATPase SERCA II. PLB, IP(3)RI, and cGMP kinase Ibeta were co-immune precipitated after expression in COS-7 cells. These results suggest that PLB may have additional functions to regulate the activity of SERCA II.

Actins↗

PubMed2003

Calcium channel alpha2delta subunits: differential expression, function, and drug binding.

Voltage-activated calcium channels are transmembrane proteins that act as transducers of electrical signals into numerous intracellular activities. On the basis of their electrophysiological properties they are classified as high- and low-voltage-activated calcium channels. High-voltage-activated calcium channels are heterooligomeric proteins consisting of a pore-forming alpha1 subunit and auxiliary alpha2delta, beta, and--in some tissues--gamma subunits. Auxiliary subunits support the membrane trafficking of the alpha1 subunit and modulate the kinetic properties of the channel. In particular, the alpha2delta subunit has been shown to modify the biophysical and pharmacological properties of the alpha1 subunit. The alpha2delta subunit is posttranslationally cleaved to form disulfide-linked alpha2 and, delta proteins, both of which are heavily glycosylated. Recently it was shown that at least four genes encode for alpha2delta subunits which are expressed in a tissue-specific manner. Their biophysical properties were characterized in coexpression studies with high- and low-voltage-activated calcium channels. Mutations in the gene encoding alpha2delta-2 have been found to underlie the ducky phenotype. This mouse mutant is a model for absence epilepsy and is characterized by spike wave seizures and cerebellar ataxia. Alpha2delta subunits can also support pharmacological interactions with drugs that are used for the treatment of epilepsy and neuropathic pain.

Acetates↗

PubMed2003

Laboratory acquired infection with recombinant vaccinia virus containing an immunomodulating construct.

Handling of Vaccinia virus represents a risk for laboratory-acquired infections, especially in individuals without completed vaccination. We report the case of a Vaccinia infection in a previously vaccinated researcher working with various genetically modified strains. We could confirm the infection by electron microscopy, positive cell culture, virus-specific PCR, sequence analysis, and viral neutralization test. The isolated virus carried a functionally inactivated cytohesin-1 gene of human origin, which had been shown to impair leukocyte adhesion by interacting with the LFA/ICAM-1 axis. The immunomodulating nature of the inserted construct might thus have added to the infectivity of the virus. We emphasize on the necessity of Vaccinia vaccination in laboratory staff working in the field.

Adjuvants, Immunologic↗

PubMed2003

Signaling through NO and cGMP-dependent protein kinases.

The gaseous molecule nitric oxide (NO) modulates a large variety of physiological functions including vascular tone, intestinal motility, platelet aggregation, proliferation, apoptosis, and neurotransmission. NO initiates diverse cellular signaling cascades which comprise nitrosylation of proteins, adenosine 5'-diphosphate (ADP)-ribosylation, or stimulation of soluble guanylyl cyclases which catalyze intracellular guanosine 3',5'-cyclic monophosphate (cGMP) synthesis. cGMP activates cGMP-dependent protein kinases (cGK) which mediate localized and global signaling. Furthermore, cGMP regulates the activity of phosphodiesterases (PDE) which modulate the duration and amplitude of cyclic nucleotide signaling. Two different types of cGK are expressed in mammals, cGKI and cGKII. Activation of the NO/cGMP/cGKI pathway induces relaxation of smooth muscle by lowering the cytosolic calcium level and/or by calcium desensitization of the contractile elements.

Calcium Signaling↗

PubMed2003

International Union of Pharmacology. XLII. Compendium of voltage-gated ion channels: cyclic nucleotide-modulated channels.

This summary article presents an overview of the molecular relationships among the voltage-gated cyclic nucleotide-modulated channels and a standard nomenclature for them, which is derived from the IUPHAR Compendium of Voltage-Gated Ion Channels. The complete Compendium, including data tables for each member of the cyclic nucleotide-modulated channel family can be found at http://www.iuphar-db.org/iuphar-ic/.

Animals↗

PubMed2003

Hypothyroidism does not affect the dihydropyridine sensitivity of precontracted murine uterus.

Thyroid hormones are known to influence various processes of cell differentiation. Recently, it was reported that hypothyroidism reduces the sensitivity to Ca2+-channel antagonists in the rat uterus. We examined the sensitivity to dihydropyridines of the uterus from mice that had reduced thyroid hormone levels. Isradipine relaxed with the same potency precontracted uterine muscle strips from control and hypothyroid mice, independently from a pseudo-pregnant state. These results demonstrate that hypothyroidism does not change dihydropyridine sensitivity (i.e., the pattern of Ca2+-channel expression) in the murine uterus.

Animals↗

PubMed2002

cGMP-mediated signaling via cGKIalpha is required for the guidance and connectivity of sensory axons.

Previous in vitro studies using cGMP or cAMP revealed a cross-talk between signaling mechanisms activated by axonal guidance receptors. However, the molecular elements modulated by cyclic nucleotides in growth cones are not well understood. cGMP is a second messenger with several distinct targets including cGMP-dependent protein kinase I (cGKI). Our studies indicated that the alpha isoform of cGKI is predominantly expressed by sensory axons during developmental stages, whereas most spinal cord neurons are negative for cGKI. Analysis of the trajectories of axons within the spinal cord showed a longitudinal guidance defect of sensory axons within the developing dorsal root entry zone in the absence of cGKI. Consequently, in cGKI-deficient mice, fewer axons grow within the dorsal funiculus of the spinal cord, and lamina-specific innervation, especially by nociceptive sensory neurons, is strongly reduced as deduced from anti-trkA staining. These axon guidance defects in cGKI-deficient mice lead to a substantial impairment in nociceptive flexion reflexes, shown using electrophysiology. In vitro studies revealed that activation of cGKI in embryonic dorsal root ganglia counteracts semaphorin 3A-induced growth cone collapse. Our studies therefore reveal that cGMP signaling is important for axonal growth in vivo and in vitro.

8-Bromo Cyclic Adenosine Monophosphate↗

PubMed2002

Pain perception in mice lacking the beta3 subunit of voltage-activated calcium channels.

The importance of voltage-activated calcium channels in pain processing has been suggested by the spinal antinociceptive action of blockers of N- and P/Q-type calcium channels as well as by gene targeting of the alpha1B subunit (N-type). The accessory beta3 subunits of calcium channels are preferentially associated with the alpha1B subunit in neurones. Here we show that deletion of the beta3 subunit by gene targeting affects strongly the pain processing of mutant mice. We pinpoint this defect in the pain-related behavior and ascending pain pathways of the spinal cord in vivo and at the level of calcium channel currents and proteins in single dorsal root ganglion neurones in vitro. The pain induced by chemical inflammation is preferentially damped by deletion of beta3 subunits, whereas responses to acute thermal and mechanical harmful stimuli are reduced moderately or not at all, respectively. The defect results in a weak wind-up of spinal cord activity during intense afferent nerve stimulation. The molecular mechanism responsible for the phenotype was traced to low expression of N-type calcium channels (alpha1B) and functional alterations of calcium channel currents in neurones projecting to the spinal cord.

Amino Acid Sequence↗

PubMed2002

L-type Ca2+ channels of the embryonic mouse heart.

In the heart, where Ca(2+) influx across the sarcolemma is essential for contraction, L-type Ca(2+) channels represent the major entry pathway of Ca(2+). Mice with a homozygous deletion of the L-type Ca(v)1.2 Ca(2+) channel gene die before day 14.5 p.c. Electrophysiological and pharmacological investigations on Ca(v)1.2-/- cardiomyocytes demonstrated that contractions depended on the influx of Ca(2+) through an L-type-like Ca(2+) channel. We analyzed now the expression pattern of various L-type Ca(2+) channels. Amplification of the alternative exons 1a and 1b revealed that embryonic cardiac cells express both Ca(v)1.2a and Ca(v)1.2b subunits. Reverse transcriptase-polymerase chain reaction (RT-PCR) amplifications indicated the expression of Ca(v)1.1 and Ca(v)1.3 in about a 1:10 ratio in Ca(v)1.2-/- embryos. Two different amino termini of the Ca(v)1.3 cDNA were found in the embryonic heart, which both gave rise to functional channels. Ca(v)1.3(1a) and Ca(v)1.3(1b) channels have similar current kinetics and voltage-dependencies as described for Ca(v)1.3(8A) channels [J. Biol. Chem. 276 (2001) 22100], but the properties of Ca(v)1.3(1a) or Ca(v)1.3(1b) channels are different from that of the L-type-like current in Ca(v)1.2-/- cardiomyocytes. The I(Ba) of Ca(v)1.3(1a) was blocked by the dihydropyridine nisoldipine with an IC(50) value of 0.13 microM at a holding potential of -80 mV. In embryonic Ca(v)1.2+/+ cardiomyocytes, I(Ba) was blocked by nisoldipine with an IC(50) value of 0.1 microM. Although the expressed Ca(v)1.3 channel has a similar affinity for nisoldipine as Ca(v)1.2+/+ cardiomyocytes, the L-type-like Ca(2+) channel found in Ca(v)1.2+/+ and -/- cardiomyocytes is not identical with the new Ca(v)1.3 splice variants.

Amino Acid Sequence↗

PubMed2002

Functional reconstitution of vascular smooth muscle cells with cGMP-dependent protein kinase I isoforms.

The cGMP-dependent protein kinase type I (cGKI) is a major mediator of NO/cGMP-induced vasorelaxation. Smooth muscle expresses two isoforms of cGKI, cGKIalpha and cGKIbeta, but the specific role of each isoform in vascular smooth muscle cells (VSMCs) is poorly understood. We have used a genetic deletion/rescue strategy to analyze the functional significance of cGKI isoforms in the regulation of the cytosolic Ca(2+) concentration by NO/cGMP in VSMCs. Cultured mouse aortic VSMCs endogenously expressed both cGKIalpha and cGKIbeta. The NO donor diethylamine NONOate (DEA-NO) and the membrane-permeable cGMP analogue 8-bromo-cGMP inhibited noradrenaline-induced Ca(2+) transients in wild-type VSMCs but not in VSMCs genetically deficient for both cGKIalpha and cGKIbeta. The defective Ca(2+) regulation in cGKI-knockout cells could be rescued by transfection of a fusion construct consisting of cGKIalpha and enhanced green fluorescent protein (EGFP) but not by a cGKIbeta-EGFP construct. Fluorescence imaging indicated that the cGKIalpha-EGFP fusion protein was concentrated in the perinuclear/endoplasmic reticulum region of live VSMCs, whereas the cGKIbeta-EGFP protein was more homogeneously distributed in the cytoplasm. These results suggest that one component of NO/cGMP-induced smooth muscle relaxation is the activation of the cGKIalpha isoform, which decreases the noradrenaline-stimulated cytosolic Ca(2+) level.

Animals↗

PubMed2002

cGMP-dependent protein kinase I mediates the negative inotropic effect of cGMP in the murine myocardium.

To study the role of cGMP-dependent protein kinase I (cGKI) for cardiac contractility, force of contraction (F(c)) was studied in electrically driven heart muscle from wild-type (WT) mice and from conventional and conditional cGKI knockout mice. Both 8-Br-cGMP and 8-pCPT-cGMP reduced Fc in cardiac muscle from juvenile WT but not from juvenile cGKI-null mutants. Similarly, the cGMP analogues reduced F(c) in forskolin-stimulated ventricular muscle from WT mice but not from cGKI-null mutants. In contrast, carbachol reduced F(c) in both groups of animals. 8-Br-cGMP reduced F(c) also in heart muscle from adult WT mice but not from adult cardiomyocyte-specific cGKI-knockout mice. These results demonstrate that cGKI mediates the negative inotropic effect of cGMP in the myocardium of juvenile and adult mice.

Animals↗

PubMed2002

Cilnidipine is a novel slow-acting blocker of vascular L-type calcium channels that does not target protein kinase C.

Cilnidipine is a novel dihydropyridine (DHP) antagonist. However, its pharmacological effects on vascular DHP-sensitive L-type channels and protein kinase C (PKC)-mediated arterial contraction is incompletely understood. To address this issue, we studied the effects of cilnidipine on multi-subunit, C-class L-type Ca2+ channels in rat aortic A7r5 cells, as well as on Ca2+ channel (L-type) alpha1C-b and (T-type) alpha1G subunits in the Xenopus oocyte expression system. Cilnidipine dose- and time-dependently inhibited Ba2+ currents in A7r5 cells, with half-maximal inhibitions (IC50) at 10 nmol/l after 10 min. Unlike classical pharmacological Ca2+ channel blockers, cilnidipine's block of Ca2+ currents did not reach steady-state levels within 10 min, indicating steady-state half-maximal inhibition of native, multi-subunit L-type channels at < 10 nmol/l. In contrast, smooth muscle alpha1Cb currents were blocked by cilnidipine at much higher doses (steady-state IC50, 20 micromol/l) whereas alpha1G currents were not inhibited by cilnidipine (30 micromol/l). Cilnidipine dose-dependently inhibited depolarization- and Ca2+-induced contractions of rat aortic rings, with an IC50 of 10 nmol/l at 10 min. However, the onset of the effects was very slow, with approximately 71% inhibition by 3 nmol/l cilnidipine after 90 min exposure to cilnidipine. In contrast, cilnidipine did not inhibit phorbol 12-myristate-13-acetate (100 nmol/l)-mediated contractions. We conclude that cilnidipine represents an extremely slow-acting DHP that targets multi-subunit L-type channels, but not PKC in arterial smooth muscle. Because cilnidipine is less potent in cells expressing the pore-forming alpha1C-b subunit, the data further suggest that this unique slow-acting mechanism of cilnidipine is mediated by a complex interaction of cilnidipine with alpha1C-b and accessory channel subunits.

Animals↗

PubMed2002

Cyclic GMP-dependent protein kinase II plays a critical role in C-type natriuretic peptide-mediated endochondral ossification.

Longitudinal bone growth is determined by endochondral ossification at the growth plate, which is located at both ends of long bones and vertebrae, and involves many systemic hormones and local regulators. C-type natriuretic peptide (CNP), a third member of the natriuretic peptide family, occurs at the growth plate and acts locally as a positive regulator of endochondral ossification through the intracellular accumulation of cyclic GMP (cGMP). The increase in cGMP concentrations is known to activate different signaling mediators, such as cyclic nucleotide phosphodiesterases, cGMP-regulated ion channels, and cGMP-dependent protein kinases (cGKs). The type II cGK (cGKII)-deficient mice (Prkg2(-/-) mice) develop dwarfism as a result of impaired endochondral ossification, suggesting that cGKII is important for the CNP-mediated endochondral ossification. However, given that Prkg2(-/-) mice differ from CNP-deficient mice (Nppc(-/-) mice) in the growth plate histology, which downstream mediator(s) of cGMP play key roles in the process is still an enigma. Here we show that targeted expression of CNP in the growth plate chondrocytes fails to rescue the skeletal defect of Prkg2(-/-) mice. Using cultured fetal mouse tibias, an in vitro model system of endochondral ossification, we also demonstrated that CNP cannot increase the longitudinal bone growth, and chondrocytic proliferation and hypertrophy, and cartilage matrix synthesis in Prkg2(-/-) mice. This study provides in vivo and in vitro genetic evidence that cGKII plays a critical role in CNP-mediated endochondral ossification.

Animals↗

PubMed2001

Regulation of cGMP-specific phosphodiesterase (PDE5) phosphorylation in smooth muscle cells.

Nitric oxide and endogenous nitrovasodilators regulate smooth muscle tone by elevation of cGMP and activation of cyclic GMP-dependent protein kinase (PKG). The amplitude and duration of the cGMP signal in smooth muscle is regulated in large part by cGMP-specific cyclic nucleotide phosphodiesterase (PDE5). Previous in vitro data have suggested that both cAMP-dependent protein kinase and PKG can regulate the activity of PDE5. To test if this type of regulation is important in the intact cell, we have generated phospho-PDE5-specific antisera and have utilized isolated smooth muscle cells from mice having a disruption in the PKG I gene as well as cells from normal human smooth muscle. The data show that in human smooth muscle cells, activation of PKG by 8-Br-cGMP led to phosphorylation and activation of PDE5. In the same cells, 8-Br-cAMP had no significant effect on PDE5 phosphorylation. Treatment of wild-type mouse aortic smooth muscle cells with 8-Br-cGMP also induced the phosphorylation of PDE5, whereas no phosphorylation was seen in smooth muscle cells isolated from mice in which the gene for PKG I had been disrupted. As with the human cells, no phosphorylation was seen in the mouse cells in response to 8-Br-cAMP. These results strongly suggest that a major regulatory pathway for control of PDE5 phosphorylation and activity in intact smooth muscle is via PKG-dependent phosphorylation of PDE5. Finally, experiments with calyculin A and okadaic acid suggest that PP1 phosphatase, the catalytic subunit of myosin phosphatase, can regulate PDE5 dephosphorylation. Together, the data suggest that phosphorylation and activation of PDE5 by PKG I and its subsequent dephosphorylation by myosin phosphatase may be key steps in the regulation of relaxation/contraction cycles of smooth muscle.

3',5'-Cyclic-GMP Phosphodiesterases↗