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Biomedical subjects

L Hein

Publications and source records attributed to L Hein.

At least 55 records · Page 3Linked to original sources
PubMed1997

Subtype-specific intracellular trafficking of alpha2-adrenergic receptors.

The three alpha2-adrenergic receptor subtypes (alpha2a, alpha2b, and alpha2c) are highly homologous G protein-coupled receptors. These receptors all couple to pertussis toxin-sensitive G proteins and have relatively similar pharmacological properties. To further explore functional differences between these receptors, we used immunocytochemical techniques to compare the ability of the three alpha2-receptor subtypes to undergo agonist-mediated internalization. The alpha2a-receptor does not internalize after agonist treatment. In contrast, we observed that the alpha2b-receptor is able to undergo agonist-induced internalization and seems to follow the same endosomal pathway used by the beta2-adrenergic receptor. Attempts to examine internalization of the alpha2c-receptor were complicated by the fact that the majority of the alpha2c receptor resides in the endoplasmic reticulum and cis/media Golgi and there is relatively little cell surface localization. Nevertheless, we were able to detect some internalization of the alpha2c-receptor after prolonged agonist treatment. However, we observed no significant movement of alpha2c-receptor from the intracellular pool to the plasma membrane during a 4-hr treatment of cells with cycloheximide, suggesting that these cells are unable to process alpha2c-receptors in the same way they process the alpha2a or alpha2b subtypes.

Amino Acid Sequence↗
PubMed1997

Intracellular trafficking of angiotensin II and its AT1 and AT2 receptors: evidence for selective sorting of receptor and ligand.

Angiotensin II (Ang II) binds to two different receptor subtypes, AT1 and AT2 receptors. In many cases, receptor stimulation by Ang II is followed by a rapid desensitization of the intracellular signal transduction and a decrease in cell surface receptor number. The present study was designed to examine by immunofluorescence microscopy the cellular trafficking pathways of Ang II and its AT1a and AT2 receptors in human embryonal kidney 293 cells stably expressing these receptor subtypes. Fluorescently labeled Ang II and AT1a receptors were rapidly internalized into endosomes. AT2 receptors were localized in the plasma membrane and did not undergo endocytosis upon agonist stimulation. After removal of agonist, AT1a receptors recycled to the plasma membrane, whereas fluorescently labeled Ang II was targeted to the lysosomal pathway. Even though no further loss of surface receptor was measurable by ligand binding at steady state, fluorescein-Ang II was continuously internalized, and cycling of receptor between endosomal vesicles and the plasma membrane was detected by antibody feeding. These experiments provide evidence for subtype-specific receptor sorting and internalization of Ang II and its AT1a receptor as a receptor-ligand complex, and they suggest that the sequestration of receptors into endosomes is in dynamic equilibrium with receptor cycling to the plasma membrane. Finally, internalization of AT1a receptors and Ang II persists after desensitization mechanisms have attenuated Ca2+ and inositol 1,4,5-trisphosphate signaling.

Ammonium Chloride↗
PubMed1997

Evidence for internalization of both type 1 angiotensin receptor subtypes (AT1a, AT1b) by a protein kinase C independent mechanism.

We and others have demonstrated the existence of two isoforms or subtypes of the angiotensin type 1 (AT1) receptor, named the AT1a and AT1b receptors. In this study we examined if both types of mouse AT1 receptors are internalized after agonist stimulation and whether protein kinase C (PKC) is involved in this process. To directly visualize the cellular localization of the receptors, an antigenic epitope was engineered onto the amino-terminal of the receptors, and stable cell lines specifically expressing each receptor subtype were isolated. Treatment of these cells with angiotensin II (Ang II) resulted in translocation of surface receptors to intracellular vesicles, together with a reduction in surface binding of Sar1Ile8-Ang II. The agonist-induced internalization of AT1a and AT1b receptors was not inhibited by the PKC inhibitor staurosporine, nor mimicked by the phorbol ester phorbol 12-myristate 13-acetate. Similar results were obtained with cultured rat vascular smooth muscle cells expressing predominantly wild-type AT1a receptors. These results suggest that both AT1a and AT1b receptors are internalized after agonist stimulation by a PKC-independent mechanism.

Angiotensin II↗
PubMed1996

Central hypotensive effects of the alpha2a-adrenergic receptor subtype.

alpha2-Adrenergic receptors (alpha2ARs) present in the brainstem decrease blood pressure and are targets for clinically effective antihypertensive drugs. The existence of three alpha2AR subtypes, the lack of subtype-specific ligands, and the cross-reactivity of alpha2AR agonists with imidazoline receptors has precluded an understanding of the role of individual alpha2AR subtypes in the hypotensive response. Gene targeting was used to introduce a point mutation into the alpha2aAR subtype in the mouse genome. The hypotensive response to alpha2AR agonists was lost in the mutant mice, demonstrating that the alpha2aAR subtype plays a principal role in this response.

Adrenergic alpha-2 Receptor Agonists↗
PubMed1996

Cardiovascular regulation in mice lacking alpha2-adrenergic receptor subtypes b and c.

alpha2-Adrenergic receptors (alpha2ARs) are essential components of the neural circuitry regulating cardiovascular function. The role of specific alpha2AR subtypes (alpha2a, alpha2b, and alpha2c) was characterized with hemodynamic measurements obtained from strains of genetically engineered mice deficient in either alpha2b or alpha2c receptors. Stimulation of alpha2b receptors in vascular smooth muscle produced hypertension and counteracted the clinically beneficial hypotensive effect of stimulating alpha2a receptors in the central nervous system. There were no hemodynamic effects produced by disruption of the alpha2c subtype. These results provide evidence for the clinical efficacy of more subtype-selective alpha2AR drugs.

Adrenergic alpha-Agonists↗
PubMed1996

Tilorone-induced lysosomal lesions: the bisbasic character of the drug is essential for its high potency to cause storage of sulphated glycosaminoglycans.

The immunomodulatory agent tilorone -2,7-bis-[2-(diethyl-amino)ethoxy]fluoren-9-one- and congeners are potent inducers of lysosomal storage of sulphated glycosaminoglycans (GAGs) in animals and cultured fibroblasts of animals and man. All potent inducers of GAG storage hitherto described are bisbasic polycyclic aromatic compounds. They are accumulated in lysosomes and disturb the degradation of GAGs, mainly dermatan sulphate. It has been proposed that the drugs cross-link the polyanionic GAG chains giving rise to undergradable drug-GAG complexes. This hypothesis implies that the bisbasic character of the drug molecules is essential for the side effect in question. In the present study, this was tested by comparing tilorone and its monobasic derivative (MT) with respect to (i) induction of GAG storage in cultured bovine corneal fibroblasts and (ii) physicochemical interactions with GAGs in vitro. The intralysosomal concentration of MT achieved after 1-3 days was of the same order of magnitude as previously shown for tilorone. Nevertheless, under conditions that did not enhance the secretion of a lysosomal enzyme (beta-hexosaminidase, EC 3.2.1.52), the ability of MT to cause storage of [35S]GAGs was significantly lower than that of tilorone. Morphological observations showed that MT was much more potent in causing lysosomal storage of polar lipids than of GAGs. CD spectroscopy with tilorone revealed that the presence of GAGs caused the primarily achiral drug molecules to display CD. This suggested a helical orientation of the tilorone molecules within GAG-drug complexes, and short intermolecular distances which allowed electronic coupling of the aromatic ring systems of adjacent drug molecules. In contrast, MT failed to display any induced optical activity, indicating the absence of highly ordered GAG-drug complexes. In conclusion, the present results show that the substitution of the planar aromatic ring system with two basic side chains is essential for the high potency of tilorone in inducing lysosomal GAG storage. This is paralleled by, and presumably causally related to, strong physicochemical interactions with GAGs.

Adjuvants, Immunologic↗
PubMed1995

Linkage mapping of the angiotensin AT2 receptor gene (Agtr2) to the mouse X chromosome.

Angiotensin II is a potent regulator of cardiovascular homeostasis and binds to two different G-protein-coupled receptors. While the type 1 receptor (AT1) mediates the cardiovascular actions of angiotensin II, the function of the recently cloned type 2 receptor (AT2) remains unknown. We have cloned the mouse AT2 receptor gene (Agtr2) and determined its map position by linkage analysis using an interspecific backcross (C57BL/6J x Mus spretus).Agtr2 is located on the proximal mouse X chromosome between DXMit85 and DXMit49, in a region of conserved synteny with a part of the human X chromosome implicated in inherited forms of premature ovarian failure. The mapping of Agtr2 may expand a region of conserved synteny with human Xq26 that includes Hprt.

Animals↗
PubMed1995

Behavioural and cardiovascular effects of disrupting the angiotensin II type-2 receptor in mice.

Angiotensin II, a potent regulator of blood pressure and of water and electrolyte balance, binds to two different G-protein-coupled receptors. The type-1 receptor (AT1) mediates the vasopressive and aldosterone-secreting effects of angiotensin II, but the function of the type-2 receptor (AT2) is unknown, although it is expressed in both adult and embryonic life. To address this question, we have generated mice lacking the gene encoding the AT2 receptor. Mutant mice develop normally, but have an impaired drinking response to water deprivation as well as a reduction in spontaneous movements. Their baseline blood pressure is normal, but they show an increased vasopressor response to injection of angiotensin II. Thus, although the AT2 receptor is not required for embryonic development, it plays a role in the central nervous system and cardiovascular functions that are mediated by the renin-angiotensin system.

Angiotensin II↗
PubMed1994

Intracellular targeting and trafficking of thrombin receptors. A novel mechanism for resensitization of a G protein-coupled receptor.

The receptor for the protease thrombin is a member of the G protein-coupled receptor family, but is activated by a unique proteolytic mechanism. The irreversibility of this proteolytic mechanism and the fact that the ligand is tethered to its receptor raise special questions about inactivation of cleaved receptors and recovery of thrombin responsiveness. We compared the intracellular trafficking of the thrombin receptor to that of the beta 2-adrenergic receptor in transfected Rat1 fibroblasts. In unstimulated cells almost all beta 2 receptors were located on the plasma membrane; by contrast, part of a cell's thrombin receptors were found in an intracellular membrane compartment which co-localized with Golgi markers. Stimulation by agonist caused internalization and subsequent recycling of the beta 2-adrenergic receptor, but most activated thrombin receptors were internalized and targeted to lysosomes. The intracellular pool of thrombin receptors found in unstimulated cells was protected from activation by thrombin, but was translocated to the plasma membrane upon activation of cell surface thrombin receptors. Replenishment of plasma membrane thrombin receptors correlated with recovery of thrombin responsiveness. These observations reveal a novel trafficking mechanism for resensitizing the thrombin receptor as opposed to the internalization/recycling pathway of other G protein-coupled receptors.

Amino Acid Sequence↗
PubMed1994

The effect of position on magnetic resonance evaluation of cerebrospinal fluid shunt function.

Since cerebrospinal fluid shunt malfunction is common and its diagnosis often requires invasive testing, we have evaluated magnetic resonance imaging (MRI) as a noninvasive test for shunt function. In prior experiments, MRI has been shown to be capable of detecting flows as low as 0.4 cc h-1 in shunt systems. Because patient movement and position in an MRI scanner are variable, we have studied the effects of shunt position on the ability to reliably detect the rate of CSF flow. Flow related enhancement of fluid flowing from 0 to 2 cc h-1 was measured in a specialized section of tubing using multiple spin echo scans (TR2000,TE20). The angle of the specialized tubing was varied relative to the axis of the MRI scanner and then the intensity of the MR signal at specific points in the tubing was measured at different flow rates. No flow standards were used as controls. The results show that the linear relationship between signal intensity and flow that had previously been demonstrated was significantly altered by changes of position. Positions angled superior to the axis of the scanner enhanced the signal while lateral changes in the plane of the scan degraded the signal. These results suggest that in order for MRI to become an accurate test for shunt function, techniques must be developed to overcome the effects of shunt position.

Cerebrospinal Fluid Shunts↗
PubMed1993

Kinetics of thrombin receptor cleavage on intact cells. Relation to signaling.

Thrombin, a protease generated at sites of vascular injury, signals cellular responses vital for hemostasis and thrombosis. How thrombin, an enzyme rather than a classical ligand, effects graded and concentration-dependent responses in its target cells has been a long-standing question. Thrombin activates its receptor by cleaving off an activation peptide to unmask a tethered peptide ligand. We utilized a thrombin receptor with an epitope-tagged activation peptide to directly demonstrate thrombin receptor cleavage and to examine the kinetics of receptor activation on intact cells. The rate of thrombin receptor cleavage was proportional to thrombin concentration over the physiologic range, but low thrombin concentrations ultimately cleaved and activated all receptors. Cumulative phosphoinositide hydrolysis in response to thrombin correlated precisely with cumulative receptor cleavage. These data strongly suggest that each cleaved and activated thrombin receptor produces a "quantum" of phosphatidylinositol hydrolysis, then shuts off. Surprisingly, this shut off occurred despite the continued presence of cleaved and "activated" receptors on the cell surface and at a time when the cells were refractory to thrombin but sensitive to agonist peptide, suggesting that a novel shut off mechanism may have evolved to deal with the tethered ligand. Unlike the case with classical ligands, cells thus cannot detect differences in thrombin concentrations as differences in fractional occupancy but rather must sense different rates of receptor activation. Because each cleaved thrombin receptor generates a quantum of second messenger, the magnitude of the cell's response to thrombin must be determined by the balance between rates of receptor activation and second messenger clearance.

Amino Acid Sequence↗
PubMed1993

The effect of nimodipine on high-energy phosphates and intracellular pH during cerebral ischemia.

Experimental and clinical studies suggest that the calcium channel blocker nimodipine may reduce cerebral ischemic injury. Using rapid acquisition phosphorus-31 nuclear magnetic resonance (31P NMR) spectroscopy, we examined the effect of nimodipine on cerebral energy metabolism during severe ischemia in gerbils. High-energy phosphates and intracellular pH were characterized at baseline and at 2-min intervals following bilateral common carotid artery (CCA) ligation. Serial forebrain spectroscopy was continued until phosphocreatine (PCr) and adenosine triphosphate (ATP) resonances disappeared. Controls (n = 10) were compared to gerbils receiving intraperitoneal nimodipine 30 min prior to carotid ligation, at the following doses: 0.5 mg/kg (n = 8), 1.0 mg/kg (n = 10), 2.0 mg/kg (n = 8), or 4.0 mg/kg (n = 4). In the control group, PCr and ATP peaks were undetectable after a mean of 5.4 +/- 0.47 min following CCA ligation. Compared with controls, the mean time for depletion of high-energy phosphates following carotid ligation was prolonged at nimodipine doses of 0.5 mg/kg and 1.0 mg/kg, but the differences did not reach statistical significance. In the 2.0 mg/kg group, however, ATP was preserved until 9.8 +/- 1.0 min following the onset of ischemia, significantly longer than the control group (p = 0.005, Mann-Whitney test). Nimodipine had no effect on the time course or severity of intracellular acidosis. In this model of severe ischemia, relatively high doses of nimodipine slowed the depletion of high-energy phosphates without altering intracellular acidosis. This suggests that nimodipine may provide cerebral protection by directly altering ischemic cellular metabolism.

Acidosis↗
PubMed1993

Heterogeneity of angiotensin synthetic pathways and receptor subtypes: physiological and pharmacological implications.

AIM: To examine the nature of multiple putative enzymatic pathways, review the evidence of angiotensin receptor heterogeneity and speculate on the implications for physiology and pharmacology. METHOD: Literature review and discussion of current research. RESULTS: New information on the renin-angiotensin system suggests that the pathways of angiotensin production are complex and that multiple receptor subtypes mediate angiotensin actions. CONCLUSIONS: The differences in structure and tissue distribution of different subtypes of angiotensin receptor may explain differences in receptor regulation in various tissues. The type 2 receptor may be primarily an embryonic receptor involved with cell differentiation and growth and may be induced in disease states. The existence of angiotensin receptor subtypes provides an opportunity for the development of receptor-specific antagonists.

Amino Acid Sequence↗
PubMed1992

Cloning, characterization, and expression of two angiotensin receptor (AT-1) isoforms from the mouse genome.

We report the existence of two structurally distinct forms of the angiotensin receptor AT-1 in the mouse. A Balb/c mouse genomic library was screened by homology screening with a polymerase chain reaction (PCR) amplified probe. Restriction mapping and sequencing of the isolated genes revealed the presence of two receptor isoforms, here named the mouse AT-1a and AT-1b receptors, containing 22 different amino acids. Receptor binding studies performed on COS-7 cells transfected with the two receptors revealed that they had similar binding profiles for angiotensin II, angiotensin III and AT-1 or AT-2 specific antagonists. Because many of the structural differences were in the carboxy terminal putative intracellular domain, we speculate that these isoforms may differ in their regulation, signal transduction, or desensitization mechanisms.

Amino Acid Sequence↗
PubMed1992

Magnetic resonance imaging analysis of extremely slow flow in a model shunt system.

Shunt malfunction is common and its diagnosis may require invasive testing that may be inaccurate or result in complications. Magnetic resonance imaging (MRI) may prove to be a useful noninvasive test of shunt function as it has been shown that MRI is capable of measuring cerebrospinal fluid (CSF) flows from 2 ml/h to 40 ml/h in model systems. Since flows in functioning shunt systems can be less than 2 ml/h, MRI must be sensitive enough to detect flow in this range in order to be a valid test for shunt function. Continuing previous studies, we have studied MRI flow-related enhancement at flow rates from 0 to 2 ml/h. Multiple spin echo scans (TR2000, TE20) were made through a specialized section of tubing in a model shunt system. The intensity of the MRI signal at points known to demonstrate maximal flow-related enhancement was measured. A linear relationship was demonstrated between signal intensity and flow as low as 0.8 ml/h. These results add support to the concept that MRI is sensitive enough to detect the lowest flows present in functioning shunt systems and therefore may be useful as a noninvasive test of shunt function.

Cerebrospinal Fluid Pressure↗
PubMed1991

An adapter for stereotactic insertion of unitized shunt systems.

When ventricles are small or distorted, or when a cyst is located deep within the brain, positioning of the proximal catheter of a shunt system may be difficult. An adapter that allows for insertion of a unitized shunt system using the Brown Roberts Wells stereotactic system is described. Using this adapter, the proximal catheter tip of the shunt system is inserted to a defined stereotactic location, eliminating the need for multiple passes when attempting to cannulate small ventricles or deep cystic lesions.

Cerebrospinal Fluid Shunts↗
PubMed1991

Differential effect of recombinant tissue plasminogen activator-induced thrombolysis in the central nervous system and systemic arteries.

Recombinant tissue plasminogen activator (rTPA) has been more effective in inducing thrombolysis in laboratory studies of intracranial thrombosis than clinical studies of systemic or coronary thrombosis would indicate. To evaluate this discrepancy, 21 rabbits were subjected to embolic occlusion of the right internal carotid artery (ICA) by retrograde injection of a tin-tagged, 2-hour-old autologous clot through the external carotid artery with angiographic monitoring in the same manner as in a previously reported model. The emboli lodged intracranially at the bifurcation of the ICA in 10 animals and in the cervical ICA in 11 animals. Digital subtraction angiography confirmed total occlusion of the ICA in all animals. Treatment started one-half hour after embolization and consisted of a bolus of 0.5 mg/kg of rTPA followed by an infusion of 1 mg/kg/h for 2 hours. All of the animals with intracranial emboli and 6 of the animals with cervical emboli were treated intravenously. The remaining 5 animals with cervical emboli were treated by intracarotid infusion of the same dose of rTPA. In the intravenously treated group, serial digital subtraction angiography documented thrombolysis in 9 of the 10 animals with intracranial emboli and only 1 of the 6 with cervical emboli. Thrombolysis occurred in 4 of the 5 rabbits with cervical emboli treated with intracarotid rTPA. Intravenous rTPA therapy produced a significantly (P less than 0.01) higher rate of thrombolysis in intracranial as compared to cervical thrombus.(ABSTRACT TRUNCATED AT 250 WORDS)

Animals↗