Biomedical subjects

S G Rasmussen

Publications and source records attributed to S G Rasmussen.

At least 19 recordsLinked to original sources

PubMed2001

Activation of the beta 2-adrenergic receptor involves disruption of an ionic lock between the cytoplasmic ends of transmembrane segments 3 and 6.

The movements of transmembrane segments (TMs) 3 and 6 at the cytoplasmic side of the membrane play an important role in the activation of G-protein-coupled receptors. Here we provide evidence for the existence of an ionic lock that constrains the relative mobility of the cytoplasmic ends of TM3 and TM6 in the inactive state of the beta(2)-adrenergic receptor. We propose that the highly conserved Arg-131(3.50) at the cytoplasmic end of TM3 interacts both with the adjacent Asp-130(3.49) and with Glu-268(6.30) at the cytoplasmic end of TM6. Such a network of ionic interactions has now been directly supported by the high-resolution structure of the inactive state of rhodopsin. We hypothesized that the network of interactions would serve to constrain the receptor in the inactive state, and the release of this ionic lock could be a key step in receptor activation. To test this hypothesis, we made charge-neutralizing mutations of Glu-268(6.30) and of Asp-130(3.49) in the beta(2)-adrenergic receptor. Alone and in combination, we observed a significant increase in basal and pindolol-stimulated cAMP accumulation in COS-7 cells transiently transfected with the mutant receptors. Moreover, based on the increased accessibility of Cys-285(6.47) in TM6, we provide evidence for a conformational rearrangement of TM6 that is highly correlated with the extent of constitutive activity of the different mutants. The present experimental data together with the recent high-resolution structure of rhodopsin suggest that ionic interactions between Asp/Glu(3.49), Arg(3.50), and Glu(6.30) may constitute a common switch governing the activation of many rhodopsin-like G-protein-coupled receptors.

Adrenergic beta-Agonists↗

PubMed2000

Agonist-induced conformational changes at the cytoplasmic side of transmembrane segment 6 in the beta 2 adrenergic receptor mapped by site-selective fluorescent labeling.

The environmentally sensitive, sulfhydryl-reactive, fluorescent probe N,N'-dimethyl-N-(iodoacetyl)-N'-(7-nitrobenz-2-oxa-1,3-diazol-4-yl) ethylene-diamine (IANBD) was used as a molecular reporter of agonist-induced conformational changes in the beta(2) adrenergic receptor, a prototype hormone-activated G protein-coupled receptor. In the background of a mutant beta(2) adrenergic receptor, with a minimal number of endogenous cysteine residues, new cysteines were introduced in positions 269(6.31), 270(6.32), 271(6.33), and 272(6.34) at the cytoplasmic side of transmembrane segment (TM) 6. The resulting mutant receptors were fully functional and bound both agonists and antagonist with high affinities also upon IANBD labeling. Fluorescence spectroscopy analysis of the purified and site-selectively IANBD-labeled mutants suggested that the covalently attached fluorophore was exposed to a less polar environment at all four positions upon agonist binding. Whereas evidence for only a minor change in the molecular environment was obtained for positions 269(6.31) and 270(6.32), the full agonist isoproterenol caused clear dose-dependent and reversible increases in fluorescence emission at positions 271(6.33) and 272(6.34). The data suggest that activation of G protein-coupled receptors, which are activated by "diffusible" ligands, involves a structural rearrangement corresponding to the cytoplasmic part of TM 6. The preferred conformations of the IANBD moiety attached to the inserted cysteines were predicted by employing a computational method that incorporated the complex hydrophobic/hydrophilic environment in which the cysteines reside. Based on these preferred conformations, it is suggested that the spectral changes reflect an agonist-promoted movement of the cytoplasmic part of TM 6 away from the receptor core and upwards toward the membrane bilayer.

Adrenergic beta-Agonists↗

PubMed2000

Biophysical characterization of the cocaine binding pocket in the serotonin transporter using a fluorescent cocaine analogue as a molecular reporter.

To explore the biophysical properties of the binding site for cocaine and related compounds in the serotonin transporter SERT, a high affinity cocaine analogue (3beta-(4-methylphenyl)tropane-2beta-carboxylic acid N-(N-methyl-N-(4-nitrobenzo-2-oxa-1,3-diazol-7-yl)ethanolamine ester hydrochloride (RTI-233); K(I) = 14 nm) that contained the environmentally sensitive fluorescent moiety 7-nitrobenzo-2-oxa-1,3-diazole (NBD) was synthesized. Specific binding of RTI-233 to the rat serotonin transporter, purified from Sf-9 insect cells, was demonstrated by the competitive inhibition of fluorescence using excess serotonin, citalopram, or RTI-55 (2beta-carbomethoxy-3beta-(4-iodophenyl)tropane). Moreover, specific binding was evidenced by measurement of steady-state fluorescence anisotropy, showing constrained mobility of bound RTI-233 relative to RTI-233 free in solution. The fluorescence of bound RTI-233 displayed an emission maximum (lambda(max)) of 532 nm, corresponding to a 4-nm blue shift as compared with the lambda(max) of RTI-233 in aqueous solution and corresponding to the lambda(max) of RTI-233 in 80% dioxane. Collisional quenching experiments revealed that the aqueous quencher potassium iodide was able to quench the fluorescence of RTI-233 in the binding pocket (K(SV =) 1.7 m(-)(1)), although not to the same extent as free RTI-233 (K(SV =) 7.2 m(-)(1)). Conversely, the hydrophobic quencher 2,2,6,6-tetramethylpiperidine-N-oxyl (TEMPO) quenched the fluorescence of bound RTI-233 more efficiently than free RTI-233. These data are consistent with a highly hydrophobic microenvironment in the binding pocket for cocaine-like uptake inhibitors. However, in contrast to what has been observed for small-molecule binding sites in, for example, G protein-coupled receptors, the bound cocaine analogue was still accessible for aqueous quenching and, thus, partially exposed to solvent.

Animals↗

PubMed2000

The effect of pH on beta(2) adrenoceptor function. Evidence for protonation-dependent activation.

The transition of rhodopsin from the inactive to the active state is associated with proton uptake at Glu(134) (1), and recent mutagenesis studies suggest that protonation of the homologous amino acid in the alpha(1B) adrenergic receptor (Asp(142)) may be involved in its mechanism of activation (2). To further explore the role of protonation in G protein-coupled receptor activation, we examined the effects of pH on the rate of ligand-induced conformational change and on receptor-mediated G protein activation for the beta(2) adrenergic receptor (beta(2)AR). The rate of agonist-induced change in the fluorescence of NBD-labeled, purified beta(2)AR was 2-fold greater at pH 6.5 than at pH 8, even though agonist affinity was lower at pH 6.5. This biophysical analysis was corroborated by functional studies; basal (agonist-independent) activation of Galpha(s) by the beta(2)AR was greater at pH 6.5 compared with pH 8.0. Taken together, these results provide evidence that protonation increases basal activity by destabilizing the inactive state of the receptor. In addition, we found that the pH sensitivity of beta(2)AR activation is not abrogated by mutation of Asp(130), which is homologous to the highly conserved acidic amino acids that link protonation to activation of rhodopsin (Glu(134)) and the alpha(1B) adrenergic receptor (Asp(142)).

Animals↗

PubMed1999

Mutation of a highly conserved aspartic acid in the beta2 adrenergic receptor: constitutive activation, structural instability, and conformational rearrangement of transmembrane segment 6.

Movements of transmembrane segments (TMs) 3 and 6 play a key role in activation of G protein-coupled receptors. However, the underlying molecular processes that govern these movements, and accordingly control receptor activation, remain unclear. To elucidate the importance of the conserved aspartic acid (Asp-130) in the Asp-Arg-Tyr motif of the beta2 adrenergic receptor (beta2AR), we mutated this residue to asparagine (D130N) to mimic its protonated state, and to alanine (D130A) to fully remove the functionality of the side chain. Both mutants displayed evidence of constitutive receptor activation. In COS-7 cells expressing either D130N or D130A, basal levels of cAMP accumulation were clearly elevated compared with cells expressing the wild-type beta2AR. Incubation of COS-7 cell membranes or purified receptor at 37 degrees C revealed also a marked structural instability of both mutant receptors, suggesting that stabilizing intramolecular constraints had been disrupted. Moreover, we obtained evidence for a conformational rearrangement by mutation of Asp-130. In D130N, a cysteine in TM 6, Cys-285, which is not accessible in the wild-type beta2AR, became accessible to methanethiosulfonate ethylammonium, a charged, sulfhydryl-reactive reagent. This is consistent with a counterclockwise rotation or tilting of TM 6 and provides for the first time structural evidence linking charge-neutralizing mutations of the aspartic acid in the DRY motif to the overall conformational state of the receptor. We propose that protonation of the aspartic acid leads to release of constraining intramolecular interactions, resulting in movements of TM 6 and, thus, conversion of the receptor to the active state.

Animals↗

PubMed1993

[Percutaneous cholecystectomy in acute cholecystitis].

Seventy-two patients with acute cholecystitis were treated with initial ultrasound-guided percutaneous transperitoneal cholecystostomy (PTCS). PTCS could be performed in all 72 patients almost without procedure related complications, and subsequent elective treatment could be done in all, except three patients where acute cholecystectomy was necessary because of accidental catheter dislocation. Seven patients died of their severe underlying illness without relationship to PTCS or cholecystitis. Eight patients with acalculous cholecystitis had no further treatment after removal of the drainage catheter. It is concluded that PTCS is a safe method for acute drainage in patients with acute cholecystitis.

Acute Disease↗

PubMed1991

[Percutaneous cholecystolithotripsy].

Thirty-two high-risk patients with severe complicating conditions and gall bladder symptoms requiring treatment, including acute cholecystitis in 28 of the patients, were treated with percutaneous cholecystolithotripsy (PTCL). This intervention was carried out under general, regional or local anaesthesia and resulted in fragmentation and removal of the stones in 90% of the patients. Cholecystectomy proved necessary in three patients on account of perforation in two and formation of "steinstrasse" in the cystic duct in one. No other complications of puncture or lithotripsy occurred and the postlithotripsy drainage time was, on the average, ten days. It is concluded that the PTCL technique is a realistic therapeutic offer to high-risk patients with gall bladder stones requiring treatment and in whom laparoscopic cholecystectomy or mini-cholecystectomy are not considered suitable.

Aged↗

PubMed1991

[Endoscopic percutaneous transhepatic cholelithotripsy in the treatment of complicated stones in the deep biliary tracts].

The results of endoscopic percutaneous transhepatic cholelithotripsy in seven patients with stones in the deep biliary passages which could not be treated by endoscopic papillotomy (EST) or extracorporeal shock wave lithotripsy (ESWL) are presented. A new dilator technique combined with mechanical or laser-lithotripsy was employed. All of the patients were relieved of the stones after uncomplicated course. It is concluded that endoscopic percutaneous cholelithotripsy which can be carried out under local anaesthesia, should be offered to patients with stones in the deep biliary passages which are not accessible to conventional treatment with EST or ESWL.

Aged↗

PubMed1991

[Extracorporeal shockwave crushing of gallstones. Preliminary report].

Extracorporeal shock wave lithotripsy (ESWL) was used for treatment of symptomatic x-ray negative stones in 23 patients. The number of ESWL sessions per patient was 1.8 (range 1-4). In 18 patients (78%), adequate fragmentation was seen comparable to results obtained elsewhere. Oral bile acid therapy was used after ESWL in the 18 patients mentioned and the mean follow-up period was five months (range 3-8 months). Four patients had by now passed all stones while 12 patients still had remaining stone fragments and one patient a gallstone. After ESWL, one patient was and one patient a gallstone. After ESWL, one patient was referred for cholecystectomy at his own request. Of the remaining five patients, cholecystectomy was performed in two and was scheduled for in three. Complications after ESWL were seen in two patients who developed acute pancreatitis. Thus, our preliminary experience shows that ESWL resulted in fragmentation and passing of gallbladder stones, but not without complications. Like the gallstone groups in Lyon, Montreal and Munich we are convinced that ESWL should be performed in accordance with prospectively designed protocols in order to establish optimal planning of indications and strategies for future treatment.

Adult↗

PubMed1991

[Percutaneous cholecystostomy and laser lithotripsy].

A case of acute cholecystitis in a high risk patient was treated in the acute phase with cholecystostomy guided by ultrasound and with introduction of a pig-tail catheter for drainage. Later, percutaneous laser lithotripsy was undertaken via a flexible choledochoscope. The recent percutaneous therapeutic principles and the possibilities in biliary calculous disease are reviewed.

Acute Disease↗

PubMed1990

Pancreatic tissue fluid pressure in chronic pancreatitis. Relation to pain, morphology, and function.

The relation between pancreatic tissue fluid pressure and pain, morphology, and function was studied in a cross-sectional investigation. Pressure measurements were performed by percutaneous fine-needle puncture. Thirty-nine patients with chronic pancreatitis were included, 25 with pain and 14 without pain. The pressure was higher in patients with pain than in patients without pain (p = 0.000001), and this was significantly related to a pain score from a visual analogue scale (p less than 0.001). Patients with pancreatic pseudocysts had both higher pressure and higher pain score than patients without (p = 0.004 and p = 0.0003, respectively). The pressure was significantly related (inversely) to pancreatic duct diameter only in the group of 19 patients with earlier pancreatic surgery (R = -0.57, p = 0.02). The pressure was not related to functional factors or the presence of pancreatic calcifications. In conclusion, pancreatic tissue fluid pressure is a valuable indicator of pain in chronic pancreatitis.

Abdominal Pain↗

PubMed1990

Evaluation of pancreatic tissue fluid pressure measurements intraoperatively and by sonographically guided fine-needle puncture.

The aim of the present study was to evaluate the needle method for pancreatic tissue fluid pressure measurements. Clinical evaluation was performed in 24 patients with chronic pancreatitis, comparing repeated pressure measurements via sonographically guided fine-needle puncture and intraoperative pressure measurements by direct puncture of pancreatic tissue and duct. In patients with chronic pancreatitis we found small week-to-week variations in sonographically guided percutaneous pressure measurements and good agreement between preoperative percutaneous pressure measurements and intraoperative pressure measurements via direct puncture. Furthermore, no significant difference was seen between pancreatic duct and tissue fluid pressure. The technical evaluation was performed by repeated pressure measurements in human pancreatic autopsy specimens and living rats in a pressure chamber at various external pressure levels. The basic calibration of the method evaluated by means of this pressure chamber study showed sufficient precision and accuracy of the needle technique for clinical and investigative purposes. In conclusion, our results suggest that pancreatic tissue fluid pressure can be reliably assessed by the needle technique.

Animals↗

PubMed1990

Evaluation of pancreatic tissue fluid pressure and pain in chronic pancreatitis. A longitudinal study.

Pancreatic tissue fluid pressure and pain were compared in a longitudinal study in nine patients undergoing drainage operations for pain in chronic pancreatitis. Pressure measurements were performed percutaneously before the operation, intraoperatively before and after the drainage procedure, and percutaneously at follow-up study 1 year after the operation. The pressures were compared with 2-week pain scores. The median predrainage pressures were increased (27 mm Hg; range, 19-34 mm Hg; normal, 7 mm Hg; range, 2-13 mm Hg). The drainage operations led to a 45% pressure decrease (range, 0-77%). At 1-year follow-up study the pressure was increased in the patients with recurrent pain, and there was a significant relation between pressure and pain (R = 0.85, p less than 0.02). Furthermore, patients with an intraoperative pressure decrease greater than 10 mm Hg had a pain-free postoperative period. The duration of the pain-free period was significantly related to the size of the intraoperative pressure decrease (R = 0.79, p less than 0.03). These results further suggest that there is a causal relationship between pancreatic tissue fluid pressure and pain in chronic pancreatitis and that the success of the drainage procedure may be predicted by intraoperative pancreatic tissue fluid pressure measurements.

Adult↗

PubMed1988