Anne L. Cahill

Histamine Evokes Greater Increases in Phosphatidylinositol Metabolism and Catecholamine Secretion in Epinephrine-Containing than in Norepinephrine-Containing Chromaffin Cells

Abstract: Chromaffin cells have H1 histamine receptors. Histamine, acting at these receptors, increases the metabolism of inositol‐containing phospholipids and stimulates catecholamine secretion from Chromaffin cells. We have investigated the effects of histamine and other agents on the accumulation of inositol monophosphate (InsP1) and catecholamine secretion in purified cultures of norepinephrine‐containing and epinephrine‐containing bovine Chromaffin cells. Histamine‐stimulated InsP, accumulation in epinephrine cells was three times greater than that in norepinephrine cells. In contrast, bradykinin caused roughly equivalent increases in InsP1 accumulation in the two Chromaffin cell subtypes. Histamine‐stimulated catecholamine secretion was also greater in epinephrine cells than in norepinephrine cells, whereas high K+, bradykinin, phorbol 12, 13‐dibutyrate, and angiotensin II all caused greater secretion from norepinephrine cells than from epinephrine cells. The density of H1 receptors in epinephrine cells was approximately three times greater than that in norepinephrine cells. The greater density of H1 receptors on epinephrine cells may account for the greater effects of histamine on InsP1 accumulation and catecholamine secretion in these cells.

Staurosporine Activates a 60,000 Mr Protein Kinase in Bovine Chromaffin Cells That Phosphorylates Myelin Basic Protein In Vitro

Abstract: Bovine chromaffin cells contain a family of renaturable protein kinases. One of these, a 60,000 Mr kinase (PK60) that phosphorylated myelin basic protein in vitro, was activated fourfold when cells were treated with the protein kinase inhibitor Staurosporine. Because staurosporine inhibits protein kinase C, the role of this kinase in the regulation of PK60 activity was investigated. Fifty nanomolar Staurosporine produced half‐maximal inhibition of protein kinase C activity in chromaffin cells, whereas ∼225 nM Staurosporine was required to induce half‐maximal activation of PK60. Other protein kinase C inhibitors, H‐7 and K‐252a, did not mimic the effect of Staurosporine on PK60 activity. Chromaffin cells have three protein kinase C isoforms: α, ε, and ζ. Prolonged treatment with phorbol esters depleted the cells of protein kinase C α and ε, but not ζ. Neither activation nor depletion of protein kinase C affected the basal activity of PK60. Moreover, Staurosporine activated PK60 in cells depleted of protein kinase C α and e; thus, Staurosporine appeared to activate PK60 by a mechanism that does not require these protein kinase C isoforms. Incubation of cell extracts with Staurosporine in vitro did not activate PK60. Incubation of these extracts with adenosine 5′‐O‐(3‐thiotriphosphate), however, caused a twofold activation of PK60. Although this suggests that PK60 activity is regulated by phosphorylation, the mechanism by which Staurosporine activates PK60 is not known. Staurosporine has been reported to promote neurite outgrowth from chromaffin cells. The role of PK60 in mediating the effects of Staurosporine on chromaffin cell function remains to be determined.

Nicotinic Agonists, Phorbol Esters, and Growth Factors Activate Two Extracellular Signal‐Regulated Kinases, ERK1 and ERK2, in Bovine Chromaffin Cells

Abstract: Treatment of bovine chromaffin cells with nicotinic agonists, phorbol esters, and growth factors increases protein kinase activity toward microtubule‐associated protein‐2 and myelin basic protein (MBP) in vitro. To characterize the kinases that are activated by these agents, we separated chromaffin cell proteins by electrophoresis in sodium dodecyl sulfate‐polyacrylamide gels into which MBP had been incorporated, allowed the proteins to renature, and then assayed MBP kinase activity by incubating the gels with [γ‐32P]ATP. Chromaffin cells contain a family of kinases that phosphorylate MBP in vitro. Two of these kinases, of Mr 46,000 and 42,000 (PK46 and PK42), were activated by treatment of the cells with dimethylphenylpiperazinium (DMPP), phorbol 12,13‐dibutyrate (PDBu), or insulin‐like growth factor I (IGF‐I). Activation of PK46 and PK42 by DMPP was dependent on extracellular Ca2+, whereas the effects of PDBu and IGF‐I were Ca2+ independent. Down‐regulation of protein kinase C by incubation of the cells with PDBu abolished the activation of PK46 and PK42 by DMPP, PDBu, and IGF‐I. Staurosporine, a protein kinase C inhibitor, prevented the activation of PK46 and PK42 by DMPP and PDBu but did not block the activation of these kinases by IGF‐I. Immunoblotting experiments with antiphosphotyrosine (anti‐PTyr) antibodies demonstrated that agents that increased the kinase activities of PK46 and PK42 also increased the apparent PTyr content of Mr 46,000 and 42,000 proteins. PK46 and PK42 comigrated with proteins that reacted with antibodies against extracellular signal‐regulated kinases (ERKs). Thus, PK46 and PK42 appear to be the bovine homologues of ERK1 and ERK2. These kinases are regulated by multiple pathways and may play a role in the mechanism by which a variety of agonists regulate chromaffin cell function.

Interaction of anesthetics with neurotransmitter release machinery proteins

General anesthetics produce anesthesia by depressing central nervous system activity. Activation of inhibitory GABA(A) receptors plays a central role in the action of many clinically relevant general anesthetics. Even so, there is growing evidence that anesthetics can act at a presynaptic locus to inhibit neurotransmitter release. Our own data identified the neurotransmitter release machinery as a target for anesthetic action. In the present study, we sought to examine the site of anesthetic action more closely. Exocytosis was stimulated by directly elevating the intracellular Ca(2+) concentration at neurotransmitter release sites, thereby bypassing anesthetic effects on channels and receptors, allowing anesthetic effects on the neurotransmitter release machinery to be examined in isolation. Three different PC12 cell lines, which had the expression of different release machinery proteins stably suppressed by RNA interference, were used in these studies. Interestingly, there was still significant neurotransmitter release when these knockdown PC12 cells were stimulated. We have previously shown that etomidate, isoflurane, and propofol all inhibited the neurotransmitter release machinery in wild-type PC12 cells. In the present study, we show that knocking down synaptotagmin I completely prevented etomidate from inhibiting neurotransmitter release. Synaptotagmin I knockdown also diminished the inhibition produced by propofol and isoflurane, but the magnitude of the effect was not as large. Knockdown of SNAP-25 and SNAP-23 expression also changed the ability of these three anesthetics to inhibit neurotransmitter release. Our results suggest that general anesthetics inhibit the neurotransmitter release machinery by interacting with multiple SNARE and SNARE-associated proteins.

Stable silencing of SNAP-25 in PC12 cells by RNA interference

BackgroundSNAP-25 is a synaptic protein known to be involved in exocytosis of synaptic vesicles in neurons and of large dense-core vesicles in neuroendocrine cells. Its role in exocytosis has been studied in SNAP-25 knockout mice, in lysed synaptosomes lacking functional SNAP-25 and in cells after treatment with botulinum toxins A or E that specifically cleave SNAP-25. These studies have shown that SNAP-25 appears to be required for most but not all evoked secretion. In order to further study the role of SNAP-25 in catecholamine secretion from PC12 cells we have used the recently developed technique of RNA interference to generate PC12 cell lines with virtually undetectable levels of SNAP-25. RNA interference is the sequence-specific silencing or knockdown of gene expression triggered by the introduction of double-stranded RNA into a cell. RNA interference can be elicited in mammalian cells in a number of ways, one of which is by the expression of small hairpin RNAs from a transfected plasmid. Selection of stably transfected cell lines expressing a small hairpin RNA allows one-time characterization of the degree and specificity of gene silencing and affords a continuing source of well-characterized knockdown cells for experimentation.ResultsA PC12 cell line stably transfected with a plasmid expressing an shRNA targeting SNAP-25 has been established. This SNAP-25 knockdown cell line has barely detectable levels of SNAP-25, but normal levels of other synaptic proteins. Catecholamine secretion elicited by depolarization of the SNAP-25 knockdown cells was reduced to 37% of control.ConclusionKnockdown of SNAP-25 in PC12 cells reduces but does not eliminate evoked secretion of catecholamines. Transient expression of human SNAP-25 in the knockdown cells rescues the deficit in catecholamine secretion.

N- and P/Q-type Ca2+ channels in adrenal chromaffin cells.

Ca2+ is the most ubiquitous second messenger found in all cells. Alterations in [Ca2+]i contribute to a wide variety of cellular responses including neurotransmitter release, muscle contraction, synaptogenesis and gene expression. Voltage‐dependent Ca2+ channels, found in all excitable cells ( Hille 1992 ), mediate the entry of Ca2+ into cells following depolarization. Ca2+ channels are composed of a large pore‐forming subunit, called the α1 subunit, and several accessory subunits. Ten different α1 subunit genes have been identified and classified into three families, Cav1‐3 ( Dunlap et al. 1995 , Catterall 2000 ). Each α1 gene produces a unique Ca2+ channel. Although chromaffin cells express several different types of Ca2+ channels, this review will focus on the Cav2.1 and Cav2.2 channels, also known as P/Q‐ and N‐type respectively ( Nowycky et al. 1985 , Llinas et al. 1989b , Wheeler et al. 1994 ). These channels exhibit physiological and pharmacological properties similar to their neuronal counterparts. N‐, P/Q and to a lesser extent R‐type Ca2+ channels are known to regulate neurotransmitter release ( Hirning et al. 1988 , Horne & Kemp 1991 , Uchitel et al. 1992 , Luebke et al. 1993 , Takahashi & Momiyama 1993 , Turner et al. 1993 , Regehr & Mintz 1994 , Wheeler et al. 1994 , Wu & Saggau 1994 , Waterman 1996 , Wright & Angus 1996 , Reid et al. 1997 ). N‐ and P/Q‐type Ca2+ channels are abundant in nerve terminals where they colocalize with synaptic vesicles. Similarly, these channels play a role in neurotransmitter release in chromaffin cells ( Garcia et al. 2006 ). N‐ and P/Q‐type channels are subject to many forms of regulation ( Ikeda & Dunlap 1999 ). This review pays particular attention to the regulation of N‐ and P/Q‐type channels by heterotrimeric G‐proteins, interaction with SNARE proteins, and channel inactivation in the context of stimulus‐secretion coupling in adrenal chromaffin cells.

Phorbol Esters Cause Preferential Secretion of Norepinephrine from Bovine Chromafin Cells

Abstract: Differential secretion of norepinephrine and epinephrine was studied in cultured bovine chromaffin cells. Nicotinic agonists and 55 mM K+ evoked a slightly greater release of norepinephrine than of epinephrine: The percentage of norepinephrine secreted was 1.5 to two times greater than the percentage of epinephrine secreted. In contrast, when the cells were treated with phorbol 12,13‐dibutyrate, the percentage of norepinephrine released was six to eight times greater than that of epinephrine released. Similar results were obtained in experiments with cultures highly enriched in either norepinephrine‐containing cells or epinephrine‐containing cells. In response to 55 mM K+, catecholamine release from norepinephrine‐containing cells was two times greater than that from epinephrine‐containing cells. In response to phorbol 12,13‐dibutyrate, secretion from norepinephrine‐containing cells was 13 times greater than that from epinephrine‐containing cells. These results suggest that protein kinase C plays a specific role in the regulation of catecholamine secretion from norepinephrine‐containing cells.

Activation of a Microtubule‐Associated Protein‐2 Kinase by Insulin‐Like Growth Factor‐I in Bovine Chromaffin Cells

Treatment of bovine chromaffin cells with insulin‐like growth factor‐I (IGF‐I) caused the activation of a protein kinase that phosphorylates microtubule‐associated protein‐2 (MAP‐2) in vitro. Activation of MAP‐2 kinase by IGF‐I varied with the time of treatment (maximal at 10–15 min) and the concentration of IGF‐I (maximal at 10 nM). The IGF‐I‐activated MAP‐2 kinase was localized to the soluble fraction of chromaffin cell extracts and required Mg2+ for activity. The IGF‐I‐activated kinase also phosphorylated myelin basic protein, but had little or no activity toward his‐tones or ribosomal S6 protein. To examine the role of protein tyrosine phosphoryiation in the activation of the MAP‐2 kinase, we isolated phosphotyrosine (PTyr)‐containing proteins from chromaffin cells by immunoaffinity adsorption on anti‐PTyr‐Sepharose beads. Anti‐PTyr‐Sepharose eluates from IGF‐I‐treated cells showed increased MAP‐2 kinase activity; thus, the MAP‐2 kinase (or a closely associated protein) appears to be a PTyr‐containing protein. Treatment of anti‐PTyr‐Sepharose eluates or crude chromaffin cell extracts with alkaline phosphatase significantly decreased kinase activity toward myelin basic protein, indicating that phosphoryiation of the IGF‐I‐activated kinase is required for its activity.

Evidence of elevated intracellular calcium levels in weaver homozygote mice.

1 A mutation in the G‐protein‐linked, inwardly rectifying K+ channel GIRK2 leads to the loss of cerebellar and dopaminergic mesencephalic neurons in weaver mice. The steps leading to cell death are not well understood but may involve constitutive influx of Na+ and Ca2+ into the neurons. 2 We found that resting [Ca2+]i was dramatically higher in cerebellar neurons from weaver mice compared to wild‐type neurons. 3 High‐K+ stimuli elicited much smaller changes in [Ca2+]i in weaver cerebellar neurons compared to wild‐type neurons. 4 weaver cerebellar granule cells could be rescued from cell death by the GIRK2wv cationic channel blocker, QX‐314. 5 QX‐314 lowered resting intracellular Ca2+ levels in weaver cerebellar granule cells. 6 These results suggest that changes in resting [Ca2+]i levels and alterations in K+ channel function are most likely to contribute to the developmental abnormalities and increased cerebellar cell death observed in weaver mice.

Differences in the composition of chromaffin granules in adrenaline and noradrenaline containing cells of bovine adrenal medulla

Several constituents of chromaffin granules were quantitatively determined in noradrenaline and adrenaline cells purified from bovine adrenal medulla. As far as secretory peptides are concerned noradrenaline granules contained slightly more secretogranin II, but much less chromogranin A than adrenaline granules. This can be explained by the dependence of the biosynthesis of chromogranin A on corticosteroids. Proteolytic processing of chromogranin A and secretogranin II was higher in noradrenaline cells which was paralleled by a higher content of the prohormone convertase PC2. Noradrenaline granules also contained a higher concentration of the vesicular monoamine transporter (vMAT2). No differences were found for dopamine beta-hydroxylase, prohormone convertase PC1, carboxypeptidase H and synaptophysin. These results indicate that the secretory cocktail of peptides released from these cells differs significantly between adrenaline and noradrenaline storing cells.