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R M Pruss

Publications and source records attributed to R M Pruss.

At least 19 recordsLinked to original sources
PubMed1999

Site-directed mutagenesis of the putative human muscarinic M2 receptor binding site.

Experimental probing of the model of the muscarinic M2 receptor binding site proposed by Hibert et al. [Hibert, M.F., Trumpp-Kallmeyer, S., Bruinsvels, A., Hoflak, K., 1991. Three-dimensional models of neurotransmitter G-binding protein-coupled receptors. Mol. Pharmacol. 40, 8-15.] was achieved by mutating each amino-acid proposed to interact with muscarinic ligands. Pharmacological analysis of the different mutant receptors transiently expressed in human embryonic kidney (HEK/293) cells was performed with a variety of agonists and antagonists. D103A, Y403A and N404A mutations prevented binding of [3H] N-methylscopolamine and [3H] quinuclidinyl benzilate with a reduction in affinity greater than 100-fold, indicating essential contributions of these residues to the binding site for the radioligands. W400A and W155A mutations had very large effects on the binding of [3H] N-methylscopolamine (150-fold, 960-fold) but modest effects on the binding of [3H] quinuclidinyl benzilate (4-fold, 17-fold). In addition, binding of oxotremorine-M, oxotremorine, arecoline and pilocarpine to W155A resulted in a greater than 100-fold decrease in affinity. Threonine mutations (T187A and T190A) alter binding of most agonists but not of antagonists. W99 makes little contribution (< 10-fold) to the binding site of the M2 receptor. D103, W155, W400, Y403 and N404 are likely to be part of the binding site for N-methylscopolamine and also to contribute to the binding site for quinuclidinyl benzilate. Some of the predicted residues do not seem to be part of the M2 receptor binding site but W155 is important for proper ligand binding on the muscarinic M2 receptor, as predicted by the proposed model.

Amino Acid Sequence↗
PubMed1997

The rat mGlu1d receptor splice variant shares functional properties with the other short isoforms of mGlu1 receptor.

Three splice variants of the rat metabotropic glutamate receptor 1 (mGlu1a, 1b and 1c receptors) have been characterized so far. All have the same sequence up to the 46th residue following the 7th transmembrane domain, followed by different carboxyl-terminal tails. Whereas mGlu1b and mGlu1c receptors possess a short intracellular carboxyl-terminal tail, the mGlu1a receptor has a very long one. Compared to cells expressing mGlu1b or mGlu1c receptors, a higher agonist potency and basal phospholipase C activity were detected in cells expressing mGlu1a receptors. Another variant with a short carboxyl-terminal tail, the HmGlu1d receptor, has been recently isolated from human brain. Here we show that the mGlu1d receptor variant also exists in the rat. Like all rat mGlu1 receptor variants, the mGlu1d receptor activates phospholipase C upon stimulation with mGlu1 receptor agonists. Although the rank order of agonist potency is the same on mGlu1a and mGlu1d receptors, agonists are less potent in stimulating phospholipase C in mGlu1d receptor-expressing cells than in cells expressing mGlu1a receptors. Moreover, like the other short variants it has no significant constitutive activity. These results indicate that the mGlu1d receptor shares similar functional properties with the other short mGlu1 receptor splice variants, and further suggests that the long carboxyl-terminal tail of the mGlu1a receptor increases phospholipase C coupling efficacy.

Amino Acid Sequence↗
PubMed1996

Human metabotropic glutamate receptor 1: mRNA distribution, chromosome localization and functional expression of two splice variants.

We have isolated clones from a human brain cDNA library coding for two splice variants of mGluR1. The combined sequences account for 6864 base pairs (bp) of the approximately 7 kilobase (kb) transcript seen on northern blots of human brain mRNA. The distribution of mGluR1 mRNA in human brain is similar to that found in rat brain and the gene for mGluR1 is localized on human chromosome 6. The mRNA for mGluR1 alpha contains an open reading frame that codes for a 1194 amino acid protein, which is slightly smaller than rat mGluR1 alpha. The major structural difference noted between the human and rat mGluR1 is a deletion of 21 nucleotides which would result in the loss of seven amino acids in the middle of a proline- and glutamine-rich region of the C-terminal tail of mGluR1 alpha. The 85 bp exon that results in the HmGluR1 beta splice variant was found to code for a protein with two amino acid differences compared to the rat receptor. Functional and pharmacological characterization of heterologously expressed human mGluR1 was performed using electrophysiological recordings from Xenopus oocytes and calcium imaging in HEK-293 cells. No major differences were found in the response of human mGluR1 to typical agonists and antagonists compared to the rat, or in the behavior of the two splice variants.

Alternative Splicing↗
PubMed1991

Agonist-activated cobalt uptake identifies divalent cation-permeable kainate receptors on neurons and glial cells.

Activation of kainate receptors causes Co2+ influx into neurons, type-2 astrocytes, and O-2A progenitor cells. Agonist-activated Co2+ uptake can be performed using cultured cells or fresh tissue slices. Based on the pattern of response to kainate, glutamate, and quisqualate, three functionally different kainate-activated ion channels (K1, K2, and K3) can be discriminated. Co2+ uptake through the K1 receptor was only activated by kainate. Both kainate and glutamate activated Co2+ uptake through the K2 receptor. Co2+ uptake through the K3 receptor was activated by all three ligands: kainate, glutamate, and quisqualate. Co2+ uptake occurred through a nonselective cation entry pathway permeable to Co2+, Ca2+, and Mn2+. The agonist-dependent activation of divalent cation influx through different kainate receptors could be correlated with expression of certain kainate receptor subunit combinations. These results are indicative of kainate receptors that may contribute to excitatory amino acid-mediated neurotoxicity.

Animals↗
PubMed1990

Agonist-dependent patterns of cytosolic Ca2+ changes in single bovine adrenal chromaffin cells: relationship to catecholamine release.

The patterns of agonist-induced elevations of cytosolic free Ca2+ ([Ca2+]i) were characterized and compared by the use of single adrenal chromaffin cells. Initial histamine- or angiotensin II (AII)-induced elevations of [Ca2+]i were equal in magnitude (peaks 329 +/- 20 [SE] and 338 +/- 46 nM, respectively). These initial increases of [Ca2+]i were transient, insensitive to either Gd3+ or removing external Ca2+, and were primarily the result of Ca2+ release from intracellular stores. After the initial peak(s) of [Ca2+]i, a second phase of moderately elevated [Ca2+]i was observed, and this response was sensitive to either Gd3+ or removing external Ca2+, supporting a role for Ca2+ entry. In most cases, the second phase of elevated [Ca2+]i was sustained during histamine stimulation but transient during AII stimulation. Maintenance of the second phase was a property of the agonist rather than of the particular cell being stimulated. Thus, individual cells exposed sequentially to histamine and AII displayed distinct patterns of [Ca2+]i changes to each agonist, regardless of the order of addition. Histamine also stimulated twice as much [3H]catecholamine release as AII, and release was completely dependent on external Ca2+. Therefore, the ability of histamine and AII to sustain (or promote) Ca2+ entry appears to underlie their efficacy as secretagogues. These data provide evidence linking agonist-dependent patterns of [Ca2+]i changes in single cells with agonist-dependent functional responses.

Adrenal Medulla↗
PubMed1990

Different patterns of agonist-stimulated increases of 3H-inositol phosphate isomers and cytosolic Ca2+ in bovine adrenal chromaffin cells: comparison of the effects of histamine and angiotensin II.

Bovine adrenal chromaffin cells (BCC) were used to compare histamine- and angiotensin II-induced changes of inositol mono-, bis-, and trisphosphate (InsP1, InsP2, and InsP3, respectively) isomers, intracellular free Ca2+ ([Ca2+]i), and the pathways of inositol phosphate metabolism. Both agonists elevated [Ca2+]i by 200 nM 3-4 s after addition, but afterwards the histamine response was much more prolonged. Histamine and angiotensin II also produced similar four- to fivefold increases of Ins(1,4,5)P3 that peaked within 5 s. Over the first minute of stimulation, however, Ins(1,4,5)P3 formation was monophasic after angiotensin II, but biphasic after histamine, evidence supporting differential regulation of angiotensin II- and histamine-stimulated signal transduction. The metabolism of Ins(1,4,5)P3 by BCC homogenates was found to proceed via (a) sequential dephosphorylation to Ins(1,4)P2 and Ins(4)P, and (b) phosphorylation to inositol 1,3,4,5-tetrakisphosphate, followed by dephosphorylation to Ins(1,3,4)P3, Ins(1,3)P2, and Ins(3,4)P2, and finally to Ins(1 or 3)P. In whole cells, Ins(1 or 3)P only increased after histamine treatment. Additionally, Ins(1,3)P2 was the only other InsP2 besides Ins(1,4)P2 to accumulate within 1 min of agonist treatment [Ins(3,4)P2 did not increase]. These results support a correlation between the time course of Ins(1,4,5)P3 formation and the time course of [Ca2+]i transients and illustrate that Ca2(+)-mobilizing agonists can produce distinguishable patterns of inositol phosphate formation and [Ca2+]i changes in BCC. Different patterns of second-messenger formation are likely to be important in signal recognition and may encode agonist-specific information.

Adrenal Glands↗
PubMed1990

Galanin gene expression in chromaffin cells is controlled by calcium and protein kinase signaling pathways.

The neuropeptide galanin (GAL) is widely distributed throughout the diffuse neuroendocrine system, and is coexpressed with acetylcholine, norepinephrine, prolactin, and a variety of other messenger substances in different cell types. Bovine chromaffin cells in primary culture synthesize and store GAL along with catecholamines, chromogranin A, and enkephalin peptides, as well as other neurosecretory products, and secrete all these molecules in response to nicotinic stimulation. The regulation of GAL biosynthesis and secretion were studied by measuring changes in messenger RNA (mRNA(GAL], and peptide immunoreactivity, 24-72 h after stimulation of secretion (40 mM potassium or 10 microM veratridine), or exposure to stimulators of protein kinase C (100 nM phorbol myristate acetate) and protein kinase A (25 microM forskolin). Depolarization-induced stimulation of GAL biosynthesis, like that of enkephalin and other neuropeptides, was calcium dependent, suggesting that calcium generally mediates both exocytotic release and new peptide synthesis thus coordinating maintenance of neuropeptide levels in chromaffin cells. GAL and mRNA(GAL) were also upregulated by stimulation of protein kinase A with forskolin. Treatment with PMA increased GAL and mRNA(GAL) to an even greater extent than depolarization. Thus GAL expression can be regulated by three distinct signal transduction systems in chromaffin cells: depolarization-stimulated calcium influx, activation of protein kinase C and activation of protein kinase A, which in addition differentially up- and down-regulate the expression of several other neurosecretory proteins and peptides resulting in different patterns of GAL/neuropeptide coexistence in bovine chromaffin cells. GAL coexistence with diverse neuroendocrine substances may reflect the relative activity of these three signalling systems in other neuroendocrine cell types as well.

Adrenal Medulla↗
PubMed1989

Dissociation of Ca2+ entry and Ca2+ mobilization responses to angiotensin II in bovine adrenal chromaffin cells.

In fura-2-loaded bovine adrenal chromaffin cells, 0.5 microM angiotensin II (AII) stimulated a 185 +/- 19 nM increase of intracellular-free calcium [( Ca2+]i) approximately 3 s after addition. The time from the onset of the response until achieving 50% recovery (t 1/2) was 67 +/- 10 s. Concomitantly, AII stimulated both the release of 45Ca2+ from prelabeled cells, and a 4-5-fold increase of [3H]inositol 1,4,5-trisphosphate [( 3H]Ins(1,4,5)P3) levels. In the presence of 50 microM LaCl3, or when extracellular-free Ca2+ [( Ca2+]o) was less than 100 nM, AII still rapidly increased [Ca2+]i by 95-135 nM, but the t 1/2 for recovery was then only 23-27 s. In medium with 1 mM MnCl2 present, AII also stimulated a small amount of Mn2+ influx, as judged by quenching of the fura-2 signal. When [Ca2+]o was normal (1.1 mM) or low (less than 60 nM), 1-2 microM ionomycin caused [Ca2+]i to increase 204 +/- 26 nM, while also releasing 45-55% of bound 45Ca2+. With low [Ca2+]o, ionomycin pretreatment abolished both the [Ca2+]i increase and 45Ca2+ release stimulated by AII. However, after ionomycin pretreatment in normal medium, AII produced a La3+-inhibitable increase of [Ca2+]i (103 +/- 13 nM) with a t 1/2 of 89 +/- 8 s, but no 45Ca2+ release. No pretreatment condition altered AII-induced formation of [3H]Ins(1,4,5)P3. We conclude that AII increased [Ca2+]i via rapid and transient Ca2+ mobilization from Ins(1,4,5)P3- and ionomycin-sensitive stores, accompanied (and/or followed) by Ca2+ entry through a La3+-inhibitable divalent cation pathway. Furthermore, the ability of AII to activate Ca2+ entry in the absence of Ca2+ mobilization (i.e. after ionomycin pretreatment) suggests a receptor-linked stimulus other than Ca2+ mobilization initiates Ca2+ entry.

Adrenal Glands↗
PubMed1988

Voltage-regulated calcium channels involved in the regulation of enkephalin synthesis are blocked by phorbol ester treatment.

Treatment of bovine chromaffin cells with 40 mM KCl stimulates a 3-fold increase in total methionine enkephalin immunoreactivity (medium plus cells) and a 4-fold increase in proenkephalin mRNA (mRNAenk). These effects of KCl, which are dependent on extracellular calcium, can be blocked by treatment with 12-O-tetradecanoylphorbol-13-acetate (TPA), although release of methionine enkephalin appears less affected. Using fura-2-loaded chromaffin cells and a dual-excitation wavelength spectrofluorometer, we have examined whether the actions of KCl and TPA on methionine enkephalin synthesis and release can be explained by changes in intracellular free calcium ([Ca2+]i). KCl produced a rapid 600 nM increase in [Ca2+]i from resting levels of approximately 170 nM. Subsequently, [Ca2+]i declined to a new steady-state plateau which was approximately 275 nM higher than the original resting levels. The postdepolarization plateau of [Ca2+]i was reduced by TPA, (-)-(R)-202,791 (a dihydropyridine calcium channel antagonist), and LaCl3 (a nonselective calcium channel blocker). TPA also inhibited potentiation of the KCl-stimulated plateau of [Ca2+]i due to (+)-(S)-202,791, a calcium channel agonist. In contrast, TPA had no effect on resting [Ca2+]i and only slightly inhibited the initial rapid KCl-stimulated increase in [Ca2+]i. The inhibitory effects were maintained for 24 h in the continuous presence of TPA. We conclude 1) that TPA inhibits enkephalin synthesis by inactivating dihydropyridine-sensitive voltage-dependent calcium channels, 2) that these channels alone maintain elevated [Ca2+]i following KCl depolarization, and 3) that sustained elevation in [Ca2+]i is necessary in order to increase enkephalin synthesis in KCl-treated chromaffin cells.

Animals↗
PubMed1988

Characterization of the altered oligosaccharide composition of the insulin receptor on neural-derived cells.

Typical insulin receptors are present on neuroblastoma cell lines. High affinity binding for insulin was present in membrane preparations from NG108 (a hybrid mouse neuroblastoma-rat glioma) as well as in membranes from SK-N-MC and SK-N-SH, two human neuroblastoma cell lines. Specific [125I]insulin binding was 24.4% for NG108, 16.9% for SK-N-MC and 5.2% for SK-N-SH at membrane protein concentrations of 0.4 mg/ml. IC50 for [125I]insulin binding was 3.4 nM in NG108 membrane preparations and 0.9 nM for SK-N-SH and 1.8 nM in SK-N-MC membranes. Apparent mol. wt. for the alpha subunits (identified by specific immunoprecipitation using the anti-insulin receptor antiserum B10) on SDS PAGE was 134 kDa for NG108; 124 kDa for SK-N-MC and 120 kDa for SK-N-SH. Neuraminidase digestion increased the mobility of the alpha subunit from both NG108 and SK-N-MC receptors to 120 kDa, whereas that from SK-N-SH were unaffected. Endoglycosidase H and endoglycosidase F digestions increased the mobility of the alpha subunits of all 3 cell lines to varying degrees, suggesting the presence of N-linked glycosylation. Insulin induced autophosphorylation of the insulin receptor beta subunit in WGA-purified membranes from all 3 cell lines. In addition, phosphorylation of a protein with an apparent mol. wt. 105 kDa was stimulated by insulin in WGA purified membranes from NG108. Tyrosine-specific kinase activity was present in the membranes from each cell line and was stimulated by insulin in a dose-dependent manner from 10(-9) to 10(-6) M. Proinsulin was about 100 times less potent in stimulating phosphorylation of the artificial substrate poly (Glu, Tyr)4:1 when compared to insulin in accordance with its lower binding affinity to the insulin receptor. Hexose transport was stimulated by insulin in all 3 cell lines. These results indicate that neuroblastoma cells contain specific insulin receptors and that they may be useful as models for studying the role of insulin in nervous tissue.

Binding, Competitive↗
PubMed1987

Monoclonal antibodies to chromaffin cells can distinguish proteins specific to or specifically excluded from chromaffin granules.

I have prepared a number of monoclonal antibodies to chromaffin cell membranes. One of these antibodies recognizes a number of antigenically related proteins that are present in all tissues examined. In the adrenal, these proteins are completely excluded from chromaffin granules but are present in other subcellular membrane fractions. This non-granule membrane-specific antibody has been designated NG3. A second antibody, CG7, binds to a single protein which segregates specifically into chromaffin granules. The protein recognized by CG7 is cytochrome b561, or chromomembrin B, one of the major protein components of chromaffin granule membranes. CG7 also labels a protein (the identical cytochrome b561) in bovine posterior pituitary neurosecretory vesicle membranes indicating that it functions in both peptidergic and catecholaminergic secretory granules. These two monoclonal antibodies provide useful probes of both granule and extra-granule membrane proteins for studies of membrane trafficking in chromaffin cells.

Adrenal Medulla↗
PubMed1987

Cytochrome b561 can be detected in many neuroendocrine tissues using a specific monoclonal antibody.

CG7 is a monoclonal antibody that recognizes cytochrome b561, a major protein component of adrenal medullary chromaffin granules. Immunocytochemical studies using this antibody have demonstrated that cytochrome b561 is present in many neural and endocrine tissues and that its distribution is correlated with the presence of either catecholamines or amidated peptides in the tissue. These tissues include neuronal cell bodies and/or fibers in the gut, blood vessels, retina, and posterior pituitary, endocrine cells of the gut, anterior and intermediate lobes of the pituitary, heart muscle, and all adrenal medullary chromaffin cells. The discovery of cytochrome b561 in many neuropeptide-containing tissues regardless of the presence of catecholamines is consistent with a general role for cytochrome b561 as a secretory granule membrane electron carrier. Its expression may be linked to an ascorbic acid requirement by both catecholamine and neuropeptide biosynthetic enzymes.

Adrenal Medulla↗
PubMed1987

Regulation of enkephalin, VIP, and chromogranin biosynthesis in actively secreting chromaffin cells. Multiple strategies for multiple peptides.

Enkephalins, vasoactive intestinal polypeptide, and chromogranin A are all contained in the secretory vesicles of chromaffin cells in culture, and are all released from this compartment by secretagogues in a calcium-dependent way. The biosynthesis of each of these peptides, however, is under quite independent regulation. The synthesis and secretion of enkephalin is tightly coupled to acetylcholine and elevated potassium stimulation by calcium influx. Once calcium enters the cell, calcium acts at pharmacologically distinct sites to elicit secretion and enhanced biosynthesis of Metenkephalin. This is demonstrated by the calcium-independent stimulation of enkephalin secretion by 1 mM barium, in contrast to the dependence on extracellular calcium of barium-stimulated biosynthesis of this peptide. The synthesis and secretion of VIP is also coupled to acetylcholine and elevated potassium stimulation by calcium influx. Treatment with barium demonstrates that calcium acts at distinct sites to stimulate secretion and biosynthesis of this peptide; however induction of VIP by barium and veratridine shows greater sensitivity to the calcium channel blocker methoxyverapamil (D600) than does the induction of Met-enkephalin by these agents. These differences in D600 sensitivity may be due to differences in calcium metabolism or voltage-dependent calcium channels in enkephalin-producing and VIP-inducible subpopulations of chromaffin cells. Chromogranin A levels are essentially unaffected by any of the agents which increase enkephalin and VIP levels, although it is secreted in parallel with enkephalins and catecholamines from chromaffin cells in response to secretagogues. We suggest that peptide hormones such as VIP and enkephalins are regulated by calcium-dependent stimulus-secretion-synthesis coupling in the chromaffin cell. Cyclic AMP is a positive regulator of enkephalin and VIP biosynthesis, but does not affect acute release of these peptides. The cAMP/protein kinase A system may be a distal mediator of peptide biosynthesis stimulated by secretagogues. Alternatively, cAMP may be involved in early developmental establishment of phenotype or long-term regulation of peptide biosynthesis by other hormones or neurotransmitters. Chromogranin A may represent a class of intravesicular, soluble proteins that are expressed constitutively by the chromaffin cell in the presence or absence of positive regulators of other systems. The biosynthesis of chromogranin A may be coupled to the production or assembly of the secretory vesicle itself.

Animals↗
PubMed1986

Calcium-activated proteolysis of neurofilament proteins in the squid giant neuron.

The phosphorylation and proteolysis of squid neurofilament proteins by endogenous kinase and calcium-activated protease activities, respectively, were studied. When axoplasm was incubated in the presence of [gamma-32P]ATP, most of the phosphate was incorporated into two neurofilament proteins: a 220-kilodalton (NF-220) and a high-molecular-weight (HMW) protein. When this phosphorylated axoplasm was subjected to endogenous calcium-activated proteolysis, two significant phosphorylated fragments were generated, i.e., a soluble 110K fragment and a pelletable 100K fragment. Immunochemical and other analyses suggest that the pelletable 100K fragment contains the common helical neurofilament rod region and that the soluble 110K protein is the putative side arm of the NF-220. In contrast, neither the HMW or the NF-220 was detected in the region of the stellate ganglion which contains the cell bodies of the giant axon. However, this region did contain a number of proteins that were sensitive to calcium-activated proteolysis and reacted with a monoclonal intermediate filament antibody. This intermediate filament antibody reacts with most of the axoplasmic proteins that copurify with neurofilaments, i.e., in the order of their intermediate filament antibody staining intensity, a 60K, 65K, 220K, and 74K protein. In the cell body preparation, the intermediate filament antibody labeled, in order of their staining intensity, a 65K, 60K, 74K, and 180K protein. In both the axoplasmic and cell body preparations, endogenous calcium-activated proteolysis generated characteristic fragments that could be labeled with the anti-intermediate filament antibody.

Animals↗
PubMed1985

Selective regulation of carboxypeptidase peptide hormone-processing enzyme during enkephalin biosynthesis in cultured bovine adrenomedullary chromaffin cells.

Bovine adrenomedullary chromaffin cells in culture were incubated with reserpine or forskolin, two agents acting through different mechanisms, which increase cellular [Met]enkephalin levels by 2-fold after 72 h. Cells were harvested and chromaffin granules were purified on a linear sucrose gradient. After reserpine treatment, carboxypeptidase-processing enzyme specific activity in chromaffin granule fractions was stimulated 1.9-fold, and Co2+-stimulated carboxypeptidase specific activity was stimulated 3-fold. The increase in enzyme activity was dependent on the time of reserpine treatment. Forskolin, on the other hand, had no significant effect on carboxypeptidase activity. The differential effects of reserpine and forskolin suggest that the carboxypeptidase-processing enzyme may be selectively regulated during periods of elevated enkephalin formation. Kinetic studies revealed that in cells exposed to reserpine, the Km value for [Met]enkephalin-Arg6 for the Co2+-stimulated carboxypeptidase activity was lowered to 0.136 from 0.447 mM, but there was no change in the Km values of the non-Co2+-stimulated carboxypeptidase activity from reserpine and control groups. Cellular levels of immunoreactive carboxypeptidase-processing enzyme, measured by a radioimmunoassay method, were not altered after reserpine treatment. These data suggest that while the total number of carboxypeptidase enzyme molecules remained constant, there may be a conversion of existing enzyme molecules to a more active form which displays a higher affinity for [Met]enkephalin-Arg6 in the presence of Co2+.

Adrenal Medulla↗
PubMed1985

Multiple populations of neuropeptide-containing cells in cultures of the bovine adrenal medulla.

Cell populations containing vasoactive intestinal polypeptide (VIP), enkephalins, and catecholamines were identified in bovine adrenal medullary cultures by immunofluorescence and radioimmunoassay. Addition of forskolin to the culture medium increased the cellular levels of both VIP and the enkephalins. These changes resulted from an increase in the number of VIP-positive cells and an increase in cellular enkephalin content.

Adrenal Medulla↗