Biomedical subjects

P Hess

Publications and source records attributed to P Hess.

At least 73 records · Page 4Linked to original sources

PubMed1993

Ion permeation through the L-type Ca2+ channel in rat phaeochromocytoma cells: two sets of ion binding sites in the pore.

1. Both inward and outward unitary Li+ currents through the L-type Ca2+ channel and discrete block of such currents by either internal or external Ca2+ are recorded. Detailed kinetic analyses are obtained for all of the four experimental configurations (internal or external Ca2+ block of either inward or outward Li+ currents). 2. No matter from which side the blocking Ca2+ ion comes, the exit (unblocking) rates are always the same at the same potential in the same direction of Li+ current flow. This indicates that the high-affinity Ca2+ binding site (the blocking site) is in the pore, and internal and external Ca2+ both go to the same site to produce the block. In other words, there can only be one high-affinity Ca2+ binding site or one set of such sites (sites separated by insignificant barriers) in the pore. Furthermore, the direction of exit of the blocking Ca2+ ion is always with, not against, the Li+ current flow. This suggests ion-ion interaction (the 'long pore effect') in the high-affinity sites. Therefore there must be more than one high-affinity site in the pore. Overall it is concluded that the pore must contain a set of high-affinity Ca2+ binding sites separated by insignificant energy barriers. 3. The voltage dependence of the off- (exit) rates is very similar in amplitude for all the four experimental configurations (e-fold change per approximately 25 mV depolarization or hyperpolarization). This strong voltage dependence in every configuration cannot be explained by any Ca2+ energy profile alone and must include a certain contribution from Li+. The mechanism underlying such a contribution seems to reside in the enhancement effect of Li+ on the exist of Ca2+. 4. The on-rates (blocking rates) for external Ca2+ are always fast no matter whether the Li+ currents are outward or inward. In certain cases the rates even approach the diffusion-controlled limit (approximately 10(9) M-1 S-1). This suggests that the high-affinity sites are very easily accessible from the outside, and probably there is no other ionic site located between the external pore mouth and the high-affinity sites. 5. The on-rates for internal Ca2+ are fast and voltage independent in outward Li+ currents, but are very slow and strongly voltage dependent in inward Li+ currents.(ABSTRACT TRUNCATED AT 400 WORDS)

Adrenal Gland Neoplasms↗

PubMed1993

Characterization of the high-affinity Ca2+ binding sites in the L-type Ca2+ channel pore in rat phaeochromocytoma cells.

1. Two major types of ion-ion interaction are demonstrated in the pore of the L-type Ca2+ channel, the 'lock-in' and the 'enhancement' effect. The former denotes that the ion at a certain site in the pore cannot move if the neighbouring site is occupied by the other ion. The latter denotes that the ion occupying a certain site may facilitate the exist of the other ion in the neighbouring site. 2. With inward currents carried by 300 mM external Li+, the exit rates of the blocking Ca2+ ion are decreased by approximately 4 times when the internal Li+ concentration is increased from 55 to 300 mM. With outward currents carried by 300 mM internal Li+, the exist rates of the blocking Ca2+ ion are decreased by approximately 2.5 times when the external Li+ concentration is increased from 55 to 300 mM. These findings demonstrate the 'lock-in' effect. 3. When inward currents are carried in Li+ in the cell-attached configuration, the on-rates of the external Ca2+ are decreased in a rectangular hyperbolic fashion with increasing external Li+ concentration (apparent dissociation constant (Kd) approximately 75 mM in activity), suggesting competition between Ca2+ and Li+ for a certain site. On the other hand, the off-rates of the blocking Ca2+ ion are increased linearly with increasing external Li+ concentration between 75 and 850 mM, and the line extrapolates to the zero point, indicating that Ca2+ exist is negligible at zero Li+. This finding demonstrates not only the existence of the enhancement effect in the channel, but also the indispensability of such an effect for Ca2+ to exit the pore. Moreover, a linear relationship up to 850 mM Li+ suggests that the affinity of Li+ to the enhancement sites is very low (apparent Kd very high) when a Ca2+ ion is present in the neighbouring site. 4. The unitary conductances of inward Ba2+ currents in the cell-attached configuration are increased with increasing Ba2+ concentration. The apparent Kd obtained from a rectangular hyperbolic fit to the data is approximately 6 mM Ba2+ (in activity). When inward Ba2+ currents are blocked by Cd2+, the on-rates of Cd2+ are decreased with increasing Ba2+ concentration also in a rectangular hyperbolic fashion, and the apparent Kd is approximately 5.5 mM. The similar Kd from these two different experiments suggests the high-affinity set can accommodate no more than two Ba2+ ions.(ABSTRACT TRUNCATED AT 400 WORDS)

Adrenal Gland Neoplasms↗

PubMed1993

Block of the L-type Ca2+ channel pore by external and internal Mg2+ in rat phaeochromocytoma cells.

1. Three to eight micromolar external Mg2+ produces discrete block of the unitary inward currents through the L-type Ca2+ channel carried by 300 mM external Li+. Like the Ca2+ block, increasing Li+ concentration decreases the Mg2+ on-rate and increases the Mg2+ off-rate. 2. These kinetic changes are saturating and the apparent dissociation constant (Kd) for the on-rates in 75 mM Li+ (in activity), the same as that in the case of Ca2+ block. This suggests that Mg2+ and Ca2+ produce the discrete block at the same site. The apparent Kd for the off-rates is 300 mM, much smaller than that in the case of Ca2+ block. This indicates that Mg2+ exerts much less repulsion on the Li+ ion in the neighbouring (enhancement) site than Ca2+, although Mg2+ and Ca2+ both have two charges. The theoretical fits to the off-rates also suggest that Mg2+ can exit the blocking sites at a rate of several hundred per second in the absence of any enhancement effect. 3. Seventeen to forty-eight micromolar internal Mg2+ produces discrete block of the outward unitary currents carried by 300 mM internal Li+. The off-rates are in general approximately 20 times faster as compared to the Mg2+ off-rates in the inward currents. This finding suggests that Mg2+ in the high-affinity sites can much more easily exit to the outside than to the inside, implying significantly higher energy barriers on the inner side of the high-affinity sites for Mg2+. 4. At least 5-10 mM internal Mg2+ is needed to produce discrete block of the inward unitary currents carried by 215 mM external Na+. The off-rates in such experiments are generally the same as those in the case of external Mg2+ block of inward currents. This suggests that internal and external Mg2+ both reach the same site, namely the high-affinity Ca2+ binding sites in the pore, to produce the discrete block. 5. Other than discrete block, 5-10 mM internal Mg2+ also decreases the size of the inward unitary current. This is most probably due to a fast block at the more internally located low-affinity sites in the pore. The fractional decrease of the currents is voltage dependent and can be fitted by a rectangular hyperbola to calculate the apparent Kd, which increases e-fold per 45 mV hyperpolarization, indicating an electrical distance of 0.3 between the low-affinity sites and the internal pore mouth.(ABSTRACT TRUNCATED AT 400 WORDS)

Adrenal Gland Neoplasms↗

PubMed1992

Evidence for cooperative interactions in potassium channel gating.

Cloning and expression of voltage-activated potassium ion-channel complementary DNAs has confirmed that these channels are composed of four identical subunits, each containing a voltage sensor. It has been generally accepted that the voltage sensors must reach a permissive state through one or more conformational ('gating') transitions before the channel can open. To test whether each subunit gates independently, we have constructed cDNAs encoding four subunits on a single polypeptide chain, enabling us to specify the subunit stoichiometry. The gating of heterotetramers made up from combinations of subunits with different gating phenotypes strongly suggests that individual subunits gate cooperatively, rather than independently. Nonindependent subunit gating is consistent with measurements of the kinetics of K(+)-channel gating currents and in line with the widespread subunit cooperativity observed in other multisubunit proteins.

Amino Acid Sequence↗

PubMed1992

Hormone withdrawal triggers a premature and sustained gene activation from delayed secondary glucocorticoid response elements.

Glucocorticoid regulatory elements, denoted GREs and delayed secondary GREs (sGREs), bind the purified glucocorticoid receptors via distinctive sequence motifs and confer a primary and delayed secondary hormone inducibility, respectively, upon a linked reporter construct in stably transfected mammalian cells. The delayed secondary responses, but not the primary responses, are preceded by a time lag of several hours and blocked by protein synthesis inhibitors. In this report, we further characterized and distinguished these hormonal inductions. A 206-base pair DNA fragment from the hepatic rat alpha 2u-globulin (RUG) gene, containing at least two delayed sGREs, was specifically activated by glucocorticoids in a dose-dependent manner via a process which is sensitive to receptor antagonist RU486. Delayed sGRE-stimulated production of correctly initiated transcripts was preceded by a time lag of 2 h, a time when the GRE-mediated induction had reached maximal levels. A pulse of glucocorticoids sustained maximal activation of the delayed secondary response but not the primary response. In fact, hormone withdrawal triggered a premature induction of this delayed secondary response, suggesting that delayed sGREs are under both negative and positive control of the hormone receptor. Two separable elements of the 206-base pair fragment, including the 29-base pair sequence of a single receptor binding site, activated the reporter expression as effectively with transient, pulsatile exposure to hormone as with continuous exposure. Our results suggest that the information content of a hormonal pulse is retained, or "memorized," more persistently by a receptor binding site of delayed sGREs than those of the prototypical GREs.

Alpha-Globulins↗

PubMed1992

A functional view of the entrances of L-type Ca2+ channels: estimates of the size and surface potential at the pore mouths.

At extreme membrane potentials, the unitary inward and outward currents through L-type Ca2+ channels become diffusion controlled and saturate. The magnitudes of these currents indicate that the pore entrances are asymmetric, with the external mouth being much larger than the internal one. On the other hand, negative surface potentials at the two ends of the pore are rather similar. Both would be significant only when the ambient ionic strength is 110 mM or less. We conclude that the surface charges will not help much in concentrating the channel's favorite divalent cations in the physiological condition. However, the pore does possess an external mouth large enough to make the important inward Ca2+ flow not limited by diffusion, even with only 1 mM external Ca2+.

Animals↗

PubMed1992

Subunit stoichiometry of a mammalian K+ channel determined by construction of multimeric cDNAs.

The subunit stoichiometry of the mammalian K+ channel KV1.1 (RCK1) was examined by linking together the coding sequences of 2-5 K+ channel subunits in a single open reading frame and tagging the expression of individual subunits with a mutation (Y379K or Y379R) that altered the sensitivity of the channel to block by external tetraethylammonium ion. Two lines of evidence argue that these constructs lead to K+ channel expression only through the formation of functional tetramers. First, currents expressed by tetrameric constructs containing a single mutant subunit have a sensitivity to tetraethylammonium that is well fitted by a single site binding isotherm. Second, a mutant subunit (Y379K) that expresses only as part of a heteromultimer contributes to the expression of functional channels when coexpressed with a trimeric construct but not a tetrameric construct.

Amino Acid Sequence↗

PubMed1992

Effects of renin-angiotensin system blockade in guinea pigs.

The goal of the present study was to compare the hemodynamic and biochemical effects of the renin inhibitor Ro 42-5892, the angiotensin converting enzyme inhibitor cilazapril, and the angiotensin II receptor blocker EXP132, the aldehyde derivative of DuP 753. The three drugs were evaluated in guinea pigs, previously treated with furosemide, using their maximal effective doses. Cilazapril decreased arterial blood pressure more than Ro 42-5892 and EXP132. In contrast, Ro 42-5892 and EXP132 had similar effects. The larger decrease of arterial pressure induced by cilazapril was not due to a larger decrease of angiotensin II in plasma and was not influenced by cyclooxygenase inhibition with indomethacin or by bradykinin antagonism with Hoe 140. After binephrectomy, most of the blood pressure-lowering effect of Ro 42-5892 disappeared. In contrast, cilazapril was still markedly effective, pointing to extrarenal effects. We conclude that in furosemide-treated guinea pigs, as opposed to previously published animal models, the decrease of arterial pressure induced by angiotensin converting enzyme inhibitors may be partly due to extrarenal effects not related to the renin-angiotensin system.

Angiotensin-Converting Enzyme Inhibitors↗

PubMed1992

[Prevention of thromboembolism in hip traumatology: low molecular weight heparin versus dextran].

A prospective, randomized trial has been undertaken to evaluate the prophylactic effects of low molecular weight heparin (LMWH) and dextran-70 in 216 patients with hip fracture during a postoperative period of ten days. Deep vein thrombosis (DVT) was diagnosed using the 125Iodine fibrinogen uptake test, confirmed by ascending venography. 113 patients received LMWH and 103 dextran-70. The frequency of DTV of 14.2% in the LMWH group was significantly lower compared with the 30.1% in the dextran group (p less than 0.003). During the first 10 days postoperative there were no fatal pulmonary embolism (PE). After this period PE occurred in 2 patients (1.8%) in the LMWH group and 1 patient (1.0%) in the dextran group. In each group one patient died from PE. There was no major bleeding in either group. The frequency of local complications was slightly higher in the dextran group (10.7%) compared with the LMWH group (3.5%). The postoperative hemoglobin level was significantly lower in dextran treated patients than in patients receiving LMWH (p less than 0.0001).

Adult↗

PubMed1992

[Can humerus shaft fractures be treated with osteosynthesis?].

The treatment of fractures of the humerus shaft is still controversial. The results of functional treatment (brace) are good, consolidation occurs in most cases within 8-10 weeks, slight malalignment can be accepted, and there is no risk of postoperative complications like osteitis, neurological iatrogenic disorders and technical errors. On the other hand a correct osteosynthesis allows painless functional postoperative treatment, the patient comfort is excellent and a selected group of patients might return to work faster. We present the results of plate fixation of humerus shaft fractures in 127 patients, operated from 1980 to 1988. 102 patients were clinically and radiologically controlled after 1 year: 85 patients (83.4%) presented an excellent or good result with complete functional recovery. 17 patients (16.6%) showed a limited range of motion in shoulder and/or elbow mostly due to other fractures of the same arm or to persistent neurological disorders (plexus or radial lesions). Postoperative complications included 2 postoperative radial palsies recovering completely within months, 5 failures of internal fixation due to technical errors, 2 pseudarthrosis and 4 postoperative infections, healed by reoperation and early removal of the implant with one exception (osteitis). We therefore conclude, that a correct plate fixation of humerus shaft fractures is an alternative to conservative treatment. The main advantage is better patient comfort and shorter disability for a selected group of patients.

Adolescent↗

PubMed1992

[Proximal femoral fractures. Is there an indication for the condylar screw (DCS)?].

Between 1983 and 1989 66 consecutive fractures of the proximal femur were treated with a condylar screw DCS. 42 patients were available for a follow-up study, 12 subtrochanteric fractures (mean age 58.5 years) and 30 intertrochanteric fractures (mean age 73 years). The primary union rate was clearly higher in the subtrochanteric group (10/12) compared to the intertrochanteric group (22/30). All the 8 implant complications (pull-out, metal fatigue) in the intertrochanteric group were associated with important posteromedial comminution in elderly patients who cannot be mobilized with only partial weight bearing postoperatively. Unstable intertrochanteric fractures in elderly patients should not be treated with the DCS. These are indications for the DHS which allows controlled telescoping. The indication for the DCS is limited to proximal shaft fractures in younger patients capable of partial weight bearing.

Adult↗

PubMed1991

Delayed secondary glucocorticoid response elements. Unusual nucleotide motifs specify glucocorticoid receptor binding to transcribed regions of alpha 2u-globulin DNA.

Glucocorticoids stimulate the transcription of rat alpha 2u-globulin (RUG) genes. Because this induction occurs after a time lag of several hours and is blocked by inhibitors of protein synthesis, it exemplifies a delayed secondary response to steroid hormones. In this report, we show that a region of RUG-transcribed DNA (approximately +1800 to +2174) contains multiple footprint sites for glucocorticoid receptor that are, apparently, organized into at least three independent binding clusters. The DNA sequences bound by the receptor and the location of binding sites were determined. A family of sequences related to half-sites of the consensus primary glucocorticoid response element (GRE) is discernible at each cluster of sites. Compared to the consensus GRE, which contains two pseudo-palindromic hexanucleotides arranged in a tail-to-tail fashion and separated by three bases, the arrangements of hexanucleotides within this segment of RUG DNA are unusual and heterogeneous. Methylation interference of a binding cluster containing three receptor footprints demonstrates that certain guanines of the GRE-like hexanucleotides are essential for efficient receptor binding. A synthetic 29-base pair (bp) RUG element, containing one receptor footprint from this cluster, selectively binds the receptor. Within this 29-bp element, six nucleotides separate two directly repeated copies of GRE-like hexanucleotides. RUG DNA fragments containing all or part of the three binding clusters, including the 29-bp element, confer a delayed secondary hormone responsiveness upon a linked heterologous promoter and reporter gene in stably transfected cell lines. We speculate that the unusual DNA sequence motifs of the receptor-binding sites are crucial for the generation of certain delayed secondary responses.

Alpha-Globulins↗

PubMed1991

Voltage-sensing residues in the S4 region of a mammalian K+ channel.

The ability of ion-channel proteins to respond to a change of the transmembrane voltage is one of the basic mechanisms underlying electrical excitability of nerve and muscle membranes. The voltage sensor has been postulated to be the fourth putative transmembrane segment (S4) of voltage-activated Na+, Ca2+ and K+ channels. Mutations of positively charged residues within S4 alter gating of Na and Shaker-type K+ channels, but quantitative correlations between the charge or a residue in S4 and the gating valence of the channel have not yet been established. Here, with improved resolution of the voltage dependence of steady-state activation, we present estimates of the equivalent gating valence with sufficient precision to allow quantitative examination of the contribution of individual charged residues to the gating valence of a mammalian non-inactivating K+ channel. We conclude that at least part of the gating charge associated with channel activation is indeed contributed by charged residues within the S4 segment.

Animals↗

PubMed1991

Pretranslational mechanisms determine the type of potassium channels expressed in the rat skeletal and cardiac muscles.

We have cloned a cDNA (RMK2) coding for a Shaker type delayed rectifier K+ channel from a rat skeletal muscle cDNA library. The clone encodes a putative protein of 602 amino acids, identical with a rat brain K+ channel Kv1 (Swanson, R., Marshall, R., Smith, J. S., Williams, J. B., Boyle, M. B., Folander, K., Luneau, C. J., Antanavage, J., Oliva, C., Burhow, S. A., Bennet, C., Stein, R. B., and Kaczmarek, L. K. (1990) Neuron 4, 929-939). Northern blot analysis showed that RMK2 is expressed in skeletal and cardiac muscle. RNase protection analysis showed that the 3'-noncoding regions of the brain, cardiac, and skeletal muscle RMK2 transcripts are identical. Cloning of the gene confirmed that the protein is encoded by a single exon (Swanson et al. (1990) Neuron 4, 929-939). We expressed RMK2 in Xenopus oocytes and showed that it encodes noninactivating delayed rectifier K+ channels, resistant to block by external tetraethylammonium, with a small unitary conductance of 8.0 picosiemens. Coinjection of RMK2 and RCK1 (RMK1) (Baumann, A., Grupe, A., Ackermann, A., and Pongs, O. (1988) EMBO J. 7, 2457-2463; Koren, G., Liman, E. R., Logothetis, D. E., Nadal-Ginard, B., and Hess, P. (1990) Neuron 4, 39-51) into Xenopus oocytes resulted in the expression of currents that have tetraethylammonium inhibition curves that differ from the linear combination of inhibition curves of the two types expressed individually. Thus, RMK2 and RCK1 (RMK1) can form heteromultimers. RNA blot hybridization analysis revealed that the RMK2 transcript is developmentally regulated in a different manner in the rat skeletal muscle, ventricle, and atrium.

Aging↗

PubMed1991

Reversible uncoupling of inactivation in N-type calcium channels.

N-type calcium channels are thought to be expressed specifically in neuronal cells and to have a dominant role in the control of neurotransmitter release from sympathetic neurons. But their unitary properties are poorly understood and the separation of neuronal Ca2+ current into components carried by N-type or L-type Ca2+ channels is controversial. Here we show that individual N-type Ca2+ channels in sympathetic neurons can carry two kinetically distinct components of current, one that is rapidly transient and one that is long lasting. The mechanism that gives rise to these two components is unexpected for Ca2+ channels: a test depolarization elicits either a rapidly inactivating, single short burst with an average duration of 40 ms, or sustained, noninactivating channel activity lasting for over 1 s. The switching between inactivating and noninactivating activity is a slow process, the occurrence of each type of unitary kinetic behaviour remaining statistically correlated over several seconds. Variable coupling of inactivation in N-type Ca2+ channels could be an effective mechanism for the modulation of neuronal excitability and synaptic plasticity.

Animals↗

PubMed1991

Ro 40-5967, in contrast to diltiazem, does not reduce left ventricular contractility in rats with chronic myocardial infarction.

Ro 40-5967 is a new calcium antagonist that binds to the same binding site as verapamil but that has been shown to have a much lesser negative inotropic effect than verapamil. The goal of the present study was to compare the effects of Ro 40-5967 and diltiazem on left ventricular contractility in vitro and in vivo in normal rats and in rats with chronic myocardial infarction induced by ligating the left coronary artery. Left ventricular contractility was assessed in vitro in isolated perfused hearts and in vivo in conscious rats by measuring left ventricular dP/dtmax + and dP/dt at P 40. In vitro, both Ro 40-5967 and diltiazem did not decrease cardiac contractility up to a dose producing complete atrioventricular block. In vivo, diltiazem decreased dP/dtmax + and dP/dt at P 40. Ro 40-5967 was less negative inotropic than diltiazem. We conclude that if these results were confirmed in clinical trials. Ro 40-5967 might be a safer drug than diltiazem, especially in patients with left ventricular dysfunction.

Animals↗

PubMed1991

Inhibition of converting enzyme and neointima formation after vascular injury in rabbits and guinea pigs.

Recently, it has been shown that cilazapril could suppress neointima formation after vascular injury in rats. The goal of the present study was to confirm these findings in guinea pigs and rabbits. Vascular injury was produced by ballooning the right carotid artery of guinea pigs and the right iliac artery of rabbits. The animals were treated with either placebo or cilazapril (30 mg/kg/day and 3 mg/kg/day in guinea pigs and rabbits, respectively). Cilazapril decreased by 42% (p less than 0.001) the neointima area in the guinea pig but was ineffective in rabbits. However, in rabbits, doses of cilazapril higher than 3 mg/kg could not be given because of known toxicological effects in the rabbit. We conclude that the protective effect of cilazapril described in rats also is observed in guinea pigs. However, in rabbits, the maximal tolerated dose of cilazapril was ineffective. These results underline the importance of ongoing clinical studies to evaluate if, in humans, cilazapril inhibits restenosis after coronary angioplasty.

Angiotensin-Converting Enzyme Inhibitors↗

PubMed1991

Effects of two angiotensin converting enzyme inhibitors and hydralazine on coronary circulation in hypertensive rats.

The goal of the present study was to compare the effects of cilazapril, a new long-acting angiotensin converting enzyme inhibitor, to those of captopril and hydralazine on the coronary vascular bed. For this purpose, spontaneously hypertensive rats were treated for 4 months with either placebo, 10 mg/kg/day cilazapril, 100 mg/kg/day captopril, or 10 mg/kg/day hydralazine. At the end of treatment, maximal coronary blood flow was measured during maximal coronary vasodilation with adenosine in isolated perfused hearts. Cilazapril was the most effective drug in increasing maximal coronary blood flow. Captopril was less effective in improving maximal coronary blood flow but was as effective as cilazapril in reducing cardiac hypertrophy. Hydralazine had an extremely small effect on cardiac hypertrophy and maximal coronary flow reserve. The ranking of efficacy was similar for the reduction of vessel wall hypertrophy in the coronary arteries and arterioles. Because of the higher efficacy of cilazapril compared with captopril, a second experiment was performed in which 10 mg/kg/day cilazapril was compared with 100 mg/kg/day captopril and 300 mg/kg/day captopril after 1 month of treatment. Captopril increased maximal coronary flow and decreased cardiac hypertrophy to the same level as cilazapril only at the highest dose. We conclude that angiotensin converting enzyme inhibitors, in contrast to hydralazine, can increase markedly maximal coronary flow in spontaneously hypertensive rats but that this increase does not always parallel the decrease of cardiac hypertrophy and is closely dose dependent.

Angiotensin-Converting Enzyme Inhibitors↗