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P A Insel

Publications and source records attributed to P A Insel.

At least 55 records · Page 3Linked to original sources
PubMed1998

Expression of altered alpha2-adrenergic phenotypic traits in normotensive humans at genetic risk of hereditary (essential) hypertension.

BACKGROUND: Essential (hereditary) hypertension is a common, though complex, trait with substantial heritability, but a still-obscure mode of inheritance. In this disorder with relatively late onset, knowledge of phenotypes with earlier penetrance would aid genetic analyses, as well as assessment of risk. OBJECTIVE: Because alpha2-adrenergic receptor alterations are among the most heritable in experimental genetic hypertension, we hypothesized enhanced expression of alpha2-adrenergic phenotypic traits in still-normotensive humans at genetic risk of hypertension. METHODS: We evaluated hemodynamic (blood pressure, cardiac output, systemic vascular resistance, stroke volume, and cardiac contractility) and biochemical (plasma drug, catecholamine, renin, and chromogranin A levels) responses to alpha2-adrenergic blockade with intravenous yohimbine in 84 normotensive subjects stratified by genetic risk of essential hypertension (67 with positive family histories and 17 with negative family histories of hypertension), as well as 18 subjects with established essential hypertension. Results were evaluated by analysis of variance, normal likelihood ratio test, and by maximum likelihood analysis for bimodality (i.e. mixtures) of response distributions. RESULTS: Blood pressure rose (P<0.001) during alpha2-adrenergic blockade, with greater response (P<0.001) in members of the hypertensive than in members of the normotensive group. Hemodynamically, the rise in blood pressure resulted from an increase in cardiac output (P<0.001), with associated increases in stroke volume (P=0.002) and cardiac contractility (P=0.006), without an overall change in systemic vascular resistance. Biochemically, plasma norepinephrine (P<0.001), epinephrine (P=0.001), and chromogranin A (P=0.02) rose, suggesting augmentation of efferent exocytotic sympathoadrenal activity. Cardiac output and stroke volume responses were correlated to increments in plasma catecholamines (especially epinephrine) for the positive group, but not for the negative group. Baseline plasma catecholamines predicted increments of stroke volume after administration of yohimbine (P=0.003-0.007) for the positive but not for the negative group. Simultaneous comparison of means and variances of cardiac output and stroke volume alpha2-adrenergic responses, by using a normal likelihood ratio test, revealed highly significant (P=0.025 to P<0.0001) differences between the groups of subjects with and without family histories of hypertension. Frequency histogram suggested that there was a bimodal distribution of responses of stroke volume to alpha2-adrenergic blockade for the normotensive group with positive family histories of hypertension; maximum likelihood analysis strongly rejected the hypothesis of a unimodal distribution, whereas the hypothesis of bimodality could not be rejected (chi2=18.4, P=0.0004). The second (exaggerated) mode of response of stroke volume to alpha2-adrenergic blockade, defined by maximum likelihood analysis, was found for 9.5% of subjects in the normotensive group with positive family histories of hypertension, and was characterized by significantly different responses of cardiac output (P=0.001), stroke volume (P<0.001), contractility (P<0.001), heart rate (P=0.03), systemic vascular resistance (P<0.001), and epinephrine (P<0.001). Even prior to alpha2-adrenergic blockade, baseline stroke volume (P=0.01), heart rate (P=0.04), systemic vascular resistance (P=0.005), and catecholamine (P=0.001-0.005) values for this subgroup were different than control values. CONCLUSIONS: We conclude that heterogeneous, bimodally distributed hemodynamic responses to alpha2-adrenergic blockade in subjects with positive family histories of hypertension suggest a discrete subgroup with early expression of perhaps Mendelian traits associated with risk of later development of hypertension. Such phenotypic traits ('intermediate phenotypes'), with earlier penetrance than hypertension itself, can be

Adolescent↗
PubMed1998

Stimulation of phospholipase D via alpha1-adrenergic receptors in Madin-Darby canine kidney cells is independent of PKCalpha and -epsilon activation.

We have demonstrated previously that protein kinase Calpha (PKCalpha) plays a key role in regulating phospholipase D (PLD) activation by nucleotides and the phorbol ester phorbol-12-myristate-13-acetate in Madin-Darby canine kidney (MDCK-D1) cells. In the current work, we investigated PLD activation in MDCK-D1 cells triggered by the adrenergic receptor agonist epinephrine and its mechanism of activation. Epinephrine, acting through the alpha1-adrenergic receptor subtype, promoted transient translocation of PKCalpha and more prolonged translocation of PKCepsilon to the membrane fraction, indicating activation of these two isoforms. In addition, epinephrine promoted activation of PLD, as shown by a sustained accumulation of phosphatidylethanol. All of these events were blocked by pretreatment of cells with the alpha1-adrenergic antagonist prazosin. D609, an inhibitor of phosphatidylcholine hydrolysis, blocked translocation of PKCalpha and PKCepsilon but did not inhibit PLD activation. Unlike results with PMA, or with the P2 purinergic receptor agonist ATP, epinephrine-stimulated PLD activity was not inhibited in MDCK-D1 cells in which PKCalpha expression is attenuated by an antisense cDNA construct or in cells in which PKC activity was inhibited by 1 microM GF 109203X. However, PLD activation by epinephrine was abolished by concomitant incubation of cells with the calcium chelator EGTA. These data, together with previous results, are consistent with the hypothesis that in MDCK-D1 cells, epinephrine acting on alpha1-adrenergic receptors, promotes a rapid increase in cytosolic Ca2+ that promotes activation of PLD through an as-yet poorly defined mechanism. The data demonstrate that different types of G protein-linked receptors that activate PLD can mediate this activation in either a PKC activation-dependent or -independent manner within a single cell type.

Adenosine Triphosphate↗
PubMed1998

Protein kinase C does not mediate phenylephrine-induced down-regulation of Madin-Darby canine kidney cell alpha-1B adrenoceptors.

We examined the down-regulation of alpha-1B adrenoceptors in Madin-Darby canine kidney D1 (MDCK) cells with an emphasis on a possible role of protein kinase C. The alpha-1 adrenoceptor agonist phenylephrine (1-100 microM) concentration-dependently down-regulated alpha-1B adrenoceptors in MDCK cells. Down-regulation by 100 microM phenylephrine was detectable after 2 hr and maximal after 8 to 24 hr. The receptor down-regulation was accompanied by a decrease in phenylephrine-stimulated inositol phosphate formation but not by an altered expression of immunodetectable Gq/11 alpha subunits. Even though alpha-1B adrenoceptor and P2 purinergic receptor stimulation promote prostaglandin E2 formation, receptor down-regulation was not prevented by indomethacin (10 microM) treatment but was partly mimicked by treatment with the purinergic receptor agonists adenosine-5'-O-(3-thio)triphosphate and 2-methylthio-ATP (300 microM each). Phorbol-12-myristate-13-acetate (1-100 nM) concentration-dependently down-regulated MDCK alpha-1B adrenoceptors to a greater extent than did phenylephrine. Three protein kinase C inhibitors, H7 (100 microM), staurosporine (100 nM) and KT5926 (1 microM), markedly attenuated receptor down-regulation promoted by phorbol ester but did not affect that by phenylephrine. Two inhibitors of Ca++/calmodulin protein kinase pathways, KT5926 (1 microM) and W-7 (30 microM), also failed to prevent phenylephrine-induced down-regulation of alpha-1B adrenoceptors. We conclude that agonist-induced down-regulation of MDCK cell alpha-1B adrenoceptors is mimicked by a protein kinase C-activating phorbol ester but that the second messenger kinases protein kinase C and Ca++/calmodulin protein kinase do not mediate agonist-induced down-regulation of the alpha-1B adrenoceptor.

1-(5-Isoquinolinesulfonyl)-2-Methylpiperazine↗
PubMed1997

Decreased transcript expression coincident with impaired glycosylation in the beta2-adrenergic receptor gene does not result from differences in the primary sequence.

Variants of the S49 mouse lymphoma cell line exhibit multiple lesions along the pathway of cyclic AMP generation in response to beta2-adrenergic stimulation. Two such variants, beta(p) and beta(d), are characterized by decreased receptor binding and mRNA expression, 50% and 25% of wild-type receptor expression, respectively. The rate of beta2-adrenergic receptor synthesis was measured and found to be decreased in the beta d cells vis-a-vis the rate in wild type cells. The molecular mass of the beta2-adrenergic receptor in the S49 wild-type, beta(p) and beta(d) variant cells was estimated by labeling the receptor with the photoaffinity probe [125I]iodocyanopindololdiazirine. Receptor size was found to be 67,000 and 47,000 Da in the wild-type and 60,000 and 42,000 in the two variant cells. This 6 kDa discrepancy in mass was abolished upon treatment of labeled cell extracts with N-glycosidase F, suggesting the possibility of either N-terminal truncation or altered glycosylation of the receptor in the variant cells. To distinguish between these possibilities, we sequenced the beta2-adrenergic receptor gene and two kilobases of the 5'-non-coding region. No differences were found in the coding region of the gene from wild-type, beta(p) and beta(d) S49 cells suggesting that both the diminished expression and the decreased size of beta2-adrenergic receptor in the beta(p) and beta(d) S49 variants are related to impaired glycosylation of the receptor. This hypothesis was substantiated by the reduced retention of the variant cells' beta2-adrenergic receptor on immobilized WGA. Furthermore, growth of the S49 cells in the presence of the alpha-mannosidase II inhibitor, swainsonine, preferentially impaired the ability of the receptors derived from the variant cells to bind to WGA. These results imply that altered expression and glycosylation of G-protein-linked receptors occur as a consequence of one or more mutations outside the receptor's open reading frame.

Amidohydrolases↗
PubMed1997

Dual role of protein kinase C in the regulation of cPLA2-mediated arachidonic acid release by P2U receptors in MDCK-D1 cells: involvement of MAP kinase-dependent and -independent pathways.

Defining the mechanism for regulation of arachidonic acid (AA) release is important for understanding cellular production of AA metabolites, such as prostaglandins and leukotrienes. Here we have investigated the differential roles of protein kinase C (PKC) and mitogen-activated protein (MAP) kinase in the regulation of cytosolic phospholipase A2 (cPLA2)-mediated AA release by P2U-purinergic receptors in MDCK-D1 cells. Treatment of cells with the P2U receptor agonists ATP and UTP increased PLA2 activity in subsequently prepared cell lysates. PLA2 activity was inhibited by the cPLA2 inhibitor AACOCF3, as was AA release in intact cells. Increased PLA2 activity was recovered in anti-cPLA2 immunoprecipitates of lysates derived from nucleotide-treated cells, and was lost from the immunodepleted lysates. Thus, cPLA2 is responsible for AA release by P2U receptors in MDCK-D1 cells. P2U receptors also activated MAP kinase. This activation was PKC-dependent since phorbol 12-myristate 13-acetate (PMA) promoted down-regulation of PKC-eliminated MAP kinase activation by ATP or UTP. Treatment of cells with the MAP kinase cascade inhibitor PD098059, the PKC inhibitor GF109203X, or down-regulation of PKC by PMA treatment, all suppressed AA release promoted by ATP or UTP, suggesting that both MAP kinase and PKC are involved in the regulation of cPLA2 by P2U receptors. Differential effects of GF109203X on cPLA2-mediated AA release and MAP kinase activation, however, were observed: at low concentrations, GF109203X inhibited AA release promoted by ATP, UTP, or PMA without affecting MAP kinase activation. Since GF109203X is more selective for PKCalpha, PKCalpha may act independently of MAP kinase to regulate cPLA2 in MDCK-D1 cells. This conclusion is further supported by data showing that PMA-promoted AA release, but not MAP kinase activation, was suppressed in cells in which PKCalpha expression was decreased by antisense transfection. Based on these data, we propose a model whereby both MAP kinase and PKC are required for cPLA2-mediated AA release by P2U receptors in MDCK-D1 cells. PKC plays a dual role in this process through the utilization of different isoforms: PKCalpha regulates cPLA2-mediated AA release independently of MAP kinase, while other PKC isoforms act through MAP kinase activation. This model contrasts with our recently demonstrated mechanism (J. Clin. Invest. 99:1302-1310.) whereby alpha1-adrenergic receptors in the same cell type regulate cPLA2-mediated AA release only through sequential activation of PKC and MAP kinase.

Animals↗
PubMed1997

Differential regulation of phospholipase D and phospholipase A2 by protein kinase C in P388D1 macrophages.

Activation of P388D1 macrophages by phorbol myristate acetate (PMA) resulted in the translocation of the protein kinase C (PKC) isoforms alpha, delta, and epsilon from the cytosol to membranes. Furthermore, PMA activated phospholipase D (PLD) in these cells, and potentiated the effect of the inflammatory lipid mediator platelet-activating factor (PAF) on PLD activation. PAF also activated phospholipase A2 (PLA2) and enhanced arachidonic acid (AA) release in P388D1 macrophages, and bacterial lipopolysaccharide (LPS) increased the responsiveness of these cells to PAF. In contrast with PLD, PLA2 activation in P388D1 macrophages was found to take place independently of PKC. This was supported by the following evidence: (i) PMA neither induced AA release nor enhanced the PAF response; (ii) inclusion of PMA along with LPS during priming did not have any effect on PAF-stimulated AA release; (iii) down-regulation of PMA-activatable PKC isoforms by chronic treatment with the phorbol ester had no effect on the PAF response; and (iv) the PKC inhibitor staurosporine did not alter the PAF-induced AA release. The present study provides an example of cells in which the direct activation of PKC by phorbol esters does not lead to a primed and/or enhanced AA release. As a unique example in which PKC activation is neither necessary nor sufficient for AA release to occur, this now allows study of the separate and distinct roles for PLD and PLA2 in signal-transduction processes. This has hitherto been difficult to achieve because of the lack of specific inhibitors of these two phospholipases.

Animals↗
PubMed1997

Role of extracellular signal-regulated kinase and PKC alpha in cytosolic PLA2 activation by bradykinin in MDCK-D1 cells.

The actions of bradykinin (BK) in Madin-Darby canine kidney (MDCK) and other cell types involve formation of arachidonic acid (AA) and AA products by as-yet-undefined mechanisms. We found that BK promoted AA release and an increase in phospholipase A2 (PLA2) activity in subsequently prepared MDCK-D1 cell lysates, both of which were Ca2+ dependent and were inhibited by the 85-kDa cytosolic PLA2 (cPLA2) inhibitor arachidonyl trifluoromethyl ketone. In addition, BK treatment of cells led to increased PLA2 activity of cPLA2 immunoprecipitated from lysates. Thus BK receptors mediate AA release via cPLA2 in MDCK-D1 cells. The BK-promoted increase of cPLA2 activity was reversed by treatment of cell lysates with potato acid phosphatase, implying that phosphorylation underlies the activation of cPLA2. However, extracellular signal-regulated kinase (ERK) appeared not to be responsible for this phosphorylation, because treatment of cells with BK (in contrast with the results obtained with epinephrine and phorbol ester) caused neither enzyme activation nor phosphorylation (as judged by molecular mass shift) of this kinase. Although the alpha isoform of protein kinase C (PKC alpha) is responsible for AA release promoted by phorbol ester treatment of MDCK-D1 cells (C. Godson, K.S. Bell, and P.A. Insel. [corrected] J. Biol. Chem. 268: 11946-11950, 1993), neither treatment of cells with the PKC alpha-selective inhibitor GF109203X nor transfection of cells with PKC alpha antisense cDNA altered BK-mediated AA release. We conclude that PKC alpha is unlikely to play an important role in the regulation of cPLA2 by BK receptors in MDCK-D1 cells. The tyrosine kinase inhibitor herbimycin A, on the other hand, inhibited both BK-promoted AA release in intact cells and cPLA2 activation in cell lysates, suggesting the involvement of tyrosine kinase in the regulation of this lipase by BK receptors. Taken together, these data suggest that BK receptors in MDCK-D1 cells regulate cPLA2 via phosphorylation mediated by kinases other than ERK and PKC alpha.

Animals↗
PubMed1996

Identification and cDNA cloning of a novel human mosaic protein, LGN, based on interaction with G alpha i2.

We have used the yeast two-hybrid system to identify proteins that interact with the alpha-subunit of the heterotrimeric GTP-binding protein, Gi2. We screened a human B cell cDNA library with full-length G alpha i2 and isolated four positive colonies, one of which expressed the 44-kDa COOH terminus of a previously unrecognized 677-amino acid (aa) protein. A full-length clone was isolated from a HeLa cell cDNA library. The deduced protein contains 10 Leu-Gly-Asn repeats, and thus we named it LGN. Computer analysis indicates that LGN is a mosaic protein with seven repeated sequences of about 40 aa in length at its N-terminal end, and four repeated sequences of about 34 aa at its C-terminal end. Each of the two repeat regions shows substantial similarity to proteins found in other organisms. RT-PCR analysis of human tissues showed that the mRNA of LGN was ubiquitously expressed. The specificity of interaction between G alpha i2 and LGN was confirmed by an in vitro binding assay using recombinant proteins. These data indicate that the yeast two-hybrid system can identify novel proteins, such as LGN, that interact with G alpha proteins. As a mosaic protein, LGN shows similarity with portions of proteins from many species and thus may define a new protein family.

Amino Acid Sequence↗
PubMed1996

Isoform-specific redistribution of protein kinase C in living cells.

We have used confocal laser scanning microscopy to determine the dynamics of distribution of activated protein kinase C (PKC) in living Madin Darby canine kidney (MDCK) cells. Using fluorescently tagged phorbol myristate acetate (PMA) as a probe for PKC we have demonstrated its distribution in association with the cell periphery and with the nucleus. Dual labeling experiments using PKC alpha and PKC beta II specific antisera indicate that activated PKC alpha is found in association with the periphery whereas activated PKC beta II is translocated to the nucleus. We have demonstrated increased activity of PKC in nuclear fractions isolated from cells treated with PMA and other PKC activators. These data indicate that upon activation individual isoforms of PKC translocate to different subcellular locations where they are likely to mediate different actions.

Animals↗
PubMed1996

The Physicians' Desk Reference. Problems and possible improvements.

The Physicians' Desk Reference (PDR) is a widely used source of drug information by American physicians and patients, but as we shall discuss, it suffers from numerous shortcomings. The PDR is a collection of written and pictorial information that is provided and paid for by pharmaceutical manufacturers. The written material for a given drug is a compilation of data and recommendations that are identical to those in the drug's package insert. The wording and directives that are included in these package inserts (and thus in the PDR) represent information that the pharmaceutical companies are permitted to present following discussion and approval by the Food and Drug Administration (FDA), Rockville, Md. The PDR is thus a negotiated effort of commercial enterprises and governmental regulators.

Aged↗
PubMed1996

Protein kinase C-dependent activation of cytosolic phospholipase A2 and mitogen-activated protein kinase by alpha 1-adrenergic receptors in Madin-Darby canine kidney cells.

We have characterized the mechanism whereby a G protein-coupled receptor, the alpha 1-adrenergic receptor, promotes cellular AA release via the activation of phospholipase A2 (PLA2) in Madin-Darby canine kidney (MDCK-D1) cells. Stimulation of cells with the receptor agonist epinephrine or with the protein kinase C (PKC) activator PMA increased AA release in intact cells and the activity of PLA2 in subsequently prepared cell lysates. The effects of epinephrine were mediated by alpha 1-adrenergic receptors since they were blocked by the alpha 1-adrenergic antagonist prazosin. Epinephrine- and PMA-promoted AA release and activation of the PLA2 were inhibited by AACOCF3, an inhibitor of the 85-kD cPLA2. The 85-kD cPLA2 could be immunoprecipitated from the cell lysate using a specific anti-cPLA2 serum. Enhanced cPLA2 activity in cells treated with epinephrine or PMA could be recovered in such immunoprecipitates, thus directly demonstrating that alpha 1-adrenergic receptors activate the 85-kD cPLA2. Activation of cPLA2 in cell lysates by PMA or epinephrine could be reversed by treatment of lysates with exogenous phosphatase. In addition, both PMA and epinephrine induced a molecular weight shift, consistent with phosphorylation, as well as an increase in activity of mitogen-activated protein (MAP) kinase. The time course of epinephrine-promoted activation of MAP kinase preceded that of the accumulation of released AA and correlated with the time course of cPLA2 activation. Down-regulation of PKC by overnight incubation of cells with PMA or inhibition of PKC with the PKC inhibitor sphingosine blocked the stimulation of MAP kinase by epinephrine and, correspondingly, epinephrine-promoted AA release was inhibited under these conditions. Similarly, blockade of MAP kinase stimulation by the MAP kinase cascade inhibitor PD098059 inhibited epinephrine-promoted AA release. The sensitivity to Ca2+ was similar, although the maximal activity of cPLA2 was enhanced by treatment of cells with epinephrine or PMA. The data thus demonstrate that in MDCK-D1 cells alpha 1-adrenergic receptors regulate AA release through phosphorylation-dependent activation of the 85-kD cPLA2 by MAP kinase subsequent to activation of PKC. This may represent a general mechanism by which G protein-coupled receptors stimulate AA release and formation of products of AA metabolism.

Animals↗
PubMed1996

P2 purinergic receptor agonists enhance cAMP production in Madin-Darby canine kidney epithelial cells via an autocrine/paracrine mechanism.

Mechanisms of cross-talk between different classes of signaling molecules are inadequately understood. We have used clonal Madin-Darby canine kidney (MDCK-D1) epithelial cells as a model system to investigate the effects of extracellular nucleotides (e.g. ATP, UTP), which promote increase in activity of several phospholipases, on cAMP production. In contrast to observations in some other cell systems, ATP and UTP, acting via P2 purinergic receptors, stimulated cAMP production in MDCK-D1 cells. At maximally effective concentrations, ATP and UTP were not additive with the beta-adrenergic receptor agonist isoproterenol, but were synergistic with forskolin in increasing cAMP production, indicating that G alpha s is activated by these nucleotides. Additionally, we found that (a) nucleotide-induced increases in cAMP were blocked by indomethacin, a cyclooxygenase inhibitor, (b) arachidonic acid increased cellular cAMP levels in an indomethacin-sensitive fashion, and (c) PGE2, the major metabolite of arachidonic acid, stimulated cAMP formation. Overall, our results suggest a mechanism by which extracellular nucleotides stimulate release of arachidonic acid which is metabolized to PGE2 which, in turn, acts in an autocrine/paracrine fashion via prostaglandin receptors to activate G alpha s and increase cAMP. Based on the ability of extracellular nucleotides to stimulate the formation and release of prostaglandins in MDCK-D1 epithelial and other cells, we hypothesize that receptor-mediated prostaglandin release may be a general mechanism that regulates cAMP formation in many types of cells.

Adenosine Triphosphate↗
PubMed1996

A key role for protein kinase A in homologous desensitization of the beta 2-adrenergic receptor pathway in S49 lymphoma cells.

We have used a [3H]forskolin binding assay to assess Gs-adenylyl cyclase interactions in intact wild-type (WT) and kin- S49 cells under conditions that desensitize the beta 2-adrenergic receptor (beta 2-AR) system. This assay provides a measurement of G alpha s-adenylyl cyclase interaction that does not rely on the determination of second messenger accumulation or enzyme activity in broken cells. Kin- S49 cells lack protein kinase A (PKA) activity and provide a unique system in which to study the relative importance of this enzyme in beta 2-AR desensitization. Although both WT and kin- S49 cells display similar kinetics of cAMP accumulation and agonist-induced cell-surface beta 2-AR loss, we found that these cell types exhibited very different extents of desensitization of forskolin binding following agonist treatment. Specifically, 10 microM isoproterenol (37 degrees C, 30 min) induced the loss of 70% of [3H]forskolin binding sites in WT cells but only 30% in kin- cells. This loss of sites in WT cells displayed a t1/2 of approximately 7 min, was agonist concentration-dependent (EC50 approximately 60 nM), was not mimicked by 8-Br-cAMP, and could be blocked by the PKA inhibitor, H89. The difference between WT and kin- cells in agonist-induced desensitization of the beta 2-AR pathway was also noted in studies of cAMP accumulation in cells. In addition, preincubation of intact cells with isoproterenol did not inhibit guanine nucleotide-dependent [3H]forskolin binding in permeabilized cells. Overall, data obtained from [3H]forskolin binding assays demonstrate the involvement of PKA in the agonist-dependent uncoupling of beta 2-AR and Gs; thus we conclude that PKA plays an important role in the homologous desensitization of the beta 2-AR-Gs-adenylyl cyclase pathway in intact cells.

Colforsin↗