Biomedical subjects

C C Felder

Publications and source records attributed to C C Felder.

At least 37 records · Page 2Linked to original sources

PubMed2000

Inflammatory cytokines enhance muscarinic-mediated arachidonic acid release through p38 mitogen-activated protein kinase in A2058 cells.

The human melanoma cell line A2058 expresses the Gq-coupled M5 subtype of muscarinic receptor. Stimulation with the cholinergic agonist, carbachol, induces a dose-dependent increase in arachidonic acid release. The carbachol-induced arachidonate release is potentiated two- to threefold by pretreatment of A2058 cells with either of the inflammatory cytokines, tumor necrosis factor-alpha or interleukin-1beta . Cytokine-induced enhancement of muscarinic-mediated arachidonic acid release peaks near 1 h. Western analysis suggests that both cytokines are capable of activating the nuclear factor-kappaB (NF-kappaB) and p38 mitogen-activated protein kinase (MAPK) pathways. Anisomycin (1 microM) treatment mimics the cytokine-induced enhancement of arachidonic acid production and activates the p38 MAPK pathway, but does not activate the NF-kappaB pathway. Furthermore, pre-treatment of A2058 cells with the putative p38 MAPK inhibitor, SB202190, ablates the cytokine-dependent augmentation without interfering with the muscarinic-mediated arachidonic acid release in untreated cells. Moreover, cytokine treatment does not affect other M5-coupled pathways (e.g., phospholipase C activity or intracellular Ca2+ mobilization), suggesting that p38 MAPK activation principally modulates muscarinic-mediated phospholipase A2 activity. Finally, in primary cultures of cells taken from rat cerebellum, key aspects of this finding are repeated in cultures enriched for glia, but not in cultures enriched for granule neurons.

Animals↗

PubMed1999

1-(1,2,5-Thiadiazol-4-yl)-4-azatricyclo[2.2.1.0(2,6)]heptanes as new potent muscarinic M1 agonists: structure-activity relationship for 3-aryl-2-propyn-1-yloxy and 3-aryl-2-propyn-1-ylthio derivatives.

Two new series of 1-(1,2,5-thiadiazol-4-yl)-4-azatricyclo[2.2.1.0(2, 6)]heptanes were synthesized and evaluated for their in vitro activity in cell lines transfected with either the human M1 or M2 receptor. 3-Phenyl-2-propyn-1-yloxy and -1-ylthio analogues substituted with halogen in the meta position showed high functional potency, efficacy, and selectivity toward the M1 receptor subtype. A quite unique functional M1 receptor selectivity was observed for compounds 8b, 8d, 8f, 9b, 9d, and 9f. Bioavailability studies in rats indicated an oral bioavailability of about 20-30%, with the N-oxide as the only detected metabolite.

Animals↗

PubMed1999

Determination of [35S]guanosine-5'-O-(3-thio)triphosphate binding mediated by cholinergic muscarinic receptors in membranes from Chinese hamster ovary cells and rat striatum using an anti-G protein scintillation proximity assay.

An assay for measuring agonist-stimulated [35S]guanosine-5'-O-(3-thio)triphosphate (GTPgamma35S) binding to heterotrimeric GTP binding proteins was developed for use in 96-well format using commercially available anti-G protein antibodies captured by anti-IgG-coated scintillation proximity assay beads. Use of an anti-Galphaq/11 antibody to measure GTPgamma35S binding mediated by M1, M3, and M5 receptors stably expressed in Chinese hamster ovary (CHO) cells resulted in a marked increase in agonist-stimulated/basal binding ratio compared with whole membrane binding. Pertussis toxin (PTX) treatment of CHO M1 cells before membrane preparation resulted in a marked reduction in agonist-stimulated GTPgamma35S binding to whole membranes. Direct coupling of M1 receptors in CHO cells to inhibitory G proteins was demonstrated using an anti-Galphai(1-3) antibody, and this binding was inhibited by 76% following PTX treatment. However, PTX had no effect on M1-mediated binding determined using anti-Galphaq/11. CHO M2 receptors mediated robust agonist-stimulated GTPgamma35S binding measured with anti-Galphai(1-3), but coupled only weakly to Galphaq/11. Using membranes from rat striatum, GTPgamma35S binding stimulated by oxotremorine M was demonstrated using anti-Galphaq/11, anti-Galphai(1-3), and anti-Galphao antibodies. Agonist-stimulated binding to striatal membranes showed a marked antibody-dependent GDP requirement with robust signals obtained using 0.1 microM GDP for anti-Galphaq/11 compared with 50 microM GDP for anti-Galphai(1-3) and anti-Galphao. The potencies observed for pirenzepine and AFDX 116 blockade of agonist-stimulated GTPgamma35S binding to striatal membranes determined with anti-Galphaq/11 and anti-Galphao suggested mediation of these responses primarily by M1 and M4 receptors, respectively. Antibody capture GTPgamma35S binding using scintillation proximity assay technology provides a convenient, productive alternative to immunoprecipitation for exploration of receptor-G protein interaction in cells and tissues.

Algorithms↗

PubMed1998

Cytosolic phospholipase A2 (cPLA2) distribution in murine brain and functional studies indicate that cPLA2 does not participate in muscarinic receptor-mediated signaling in neurons.

Cytosolic phospholipase A2 (cPLA2) catalyzes the selective release of arachidonic acid from the sn-2 position of membrane phospholipids and has been suggested as an effector in the receptor-mediated release of arachidonic acid in signal transduction. The potential role of cPLA2 as an effector in muscarinic acetylcholine receptor signaling was investigated through ectopic expression of either the m1 or m5 receptor in combination with cPLA2 in COS-1, CHO and U-373 MG cell lines. U-373 MG and COS-1 cells express undetectable or very low levels of cPLA2. CHO cell extracts are characterized by a significant endogenous PLA2 activity that was increased over 20-fold following transient expression with cPLA2 cDNA. However, in none of the cells lines did the co-expression of muscarinic receptor and cPLA2 result in a significant increase in muscarinic receptor-mediated arachidonic acid release over cells expressing muscarinic receptor alone. The distribution of cPLA2 mRNA and cPLA2 immunoreactivity in murine brain were determined in order to investigate a potential role for cPLA2 in neurotransmission. cPLA2 mRNA was expressed in white matter, including cells contained within linear arrays characteristic of interfascicular oligodendrocytes. cPLA2 immunoreactivity in white matter was evident throughout the processes of fibrous astrocytes. cPLA2 expression in gray matter was confined to astrocytes at the pial surface of the brain. cPLA2 mRNA was detected in pia mater, both at the brain surface and inner core of the choroid plexus. cPLA2 may not be directly linked to neurotransmission since enzyme expression, mRNA, and cPLA2 immunoreactivity were undetectable in neurons of murine brain. Support or regulation of neurotransmission may be provided through the activity of cPLA2 in glial cells.

Animals↗

PubMed1998

Cannabinoid receptors and their endogenous agonist, anandamide.

Cannabinoids are a class of compound found in marijuana which have been known for their therapeutic and psychoactive properties for at least 4000 years. Isolation of the active principle in marijuana, delta9-THC, provided the lead structure in the development of highly potent congeners which were used to probe for the mechanism of marijuana action. Cannabinoids were shown to bind to selective binding sites in brain tissue thereby regulating second messenger formation. Such studies led to the cloning of three cannabinoid receptor subtypes, CB1, CB2, and CB1A all of which belong to the superfamily of G protein-coupled plasma membrane receptors. Analogous to the discovery of endogenous opiates, isolation of cannabinoid receptors provided the appropriate tool to isolate an endogenous cannabimimetic eicosanoid, anandamide, from porcine brain. Recent studies indicate that anandamide is a member of a family of fatty acid ethanolamides that may represent a novel class of lipid neurotransmitters. This review discusses recent progress in cannabinoid research with a focus on the receptors for delta9-THC, their coupling to second messenger responses, and the endogenous lipid cannabimimetic, anandamide.

Animals↗

PubMed1998

Cannabinoid receptors and their endogenous agonists.

Marijuana has been in use for over 4000 years as a therapeutic and as a recreational drug. Within the past decade, two cannabinoid receptor types have been identified, their signal transduction characterized, and an endogenous lipid agonist isolated from mammalian tissues. The CB1 cannabinoid receptor is widely distributed in mammalian tissues, with the highest concentrations found in brain neurons. CB1 receptors are coupled to modulation of adenylate cyclase and ion channels. The CB2 receptor is found in cells of the immune system and is coupled to inhibition of adenylate cyclase. Both receptor types selectively bind delta 9-THC, the active principle in marijuana, and anandamide (arachidonylethanolamide), an endogenous cannabimimetic eicosanoid. Progress is being made in the development of novel agonists and antagonists with receptor subtype selectivity, mice with genetic deletion of the cannabinoid receptors, and receptor-specific antibodies, which should help in providing a better understanding of the physiological role of the cannabinoid receptors.

Animals↗

PubMed1998

LY320135, a novel cannabinoid CB1 receptor antagonist, unmasks coupling of the CB1 receptor to stimulation of cAMP accumulation.

LY320135 is a selective antagonist for the brain CB1 receptor, having greater than 70-fold higher affinity for the CB1 than the peripheral CB2 receptor. The Ki values for LY320135 at the CB1 and CB2 receptors, transfected and stably expressed in cell lines, were 224 nM and > 10 microM, respectively. Similar Ki values were measured in binding studies performed on cerebellum and spleen membrane preparations endogenously expressing the CB1 (203 nM) and CB2 (> 10 microM) receptors, respectively. LY320135 functionally reversed anandamide-mediated adenylate cyclase inhibition in Chinese hamster ovary (CHO) cells stably expressing the CB1 receptor. Pertussis toxin treatment of CHO cells expressing the CB1 receptor attenuated the anandamide-mediated inhibition of adenylate cyclase and unmasked a stimulatory effect of anandamide on adenylate cyclase. The stimulatory component was blocked with LY320135. This compound also blocked WIN 55212-2-mediated inhibition of N-type calcium channels and activation of inwardly rectifying potassium channels in N18 and AtT-20-CB2 cells, respectively. LY320135 is a promising lead compound for the further development of novel, potent and selective cannabinoid antagonists of novel structure.

Animals↗

PubMed1997

A9 fibroblasts transfected with the m3 muscarinic receptor clone express a Ca2+ channel activated by carbachol, GTP and GDP.

Muscarinic m3 receptor-mediated changes in cytosolic Ca2+ concentration ([Ca2+]i) occur by activation of Ca2+ release channels present in the endoplasmic reticulum membrane and Ca2+ entry pathways across the plasma membrane. In this report we demonstrate the coupling of m3 muscarinic receptors to the activation of a voltage-insensitive, cation-selective channel of low conductance (3.2 +/- 0.6 pS; 25 mM Ca2+ as charge carrier) in a fibroblast cell line expressing m3 muscarinic receptor clone (A9m3 cells). Carbachol (CCh)-induced activation of the cation-selective channel occurred both in whole cell and excised membrane patches (CCh on the external side), suggesting that the underlying mechanism involves receptor-channel coupling independent of intracellular messengers. In excised inside-out membrane patches from nonstimulated A9m3 cells GTP (10 microM) and GDP (10 microM) activated cation-selective channels with conductances of approximately 4.3 and 3.3 pS, (25 mM Ca2+ as charge carrier) respectively. In contrast, ATP (10 microM), UTP (10 microM) or CTP (10 microM) failed to activate the channel. Taken together, these results suggest that carbachol and guanine nucleotides regulate the activation of a cation channel that conducts calcium.

Animals↗

PubMed1997

Concurrent stimulation of cannabinoid CB1 and dopamine D2 receptors augments cAMP accumulation in striatal neurons: evidence for a Gs linkage to the CB1 receptor.

Cannabinoids act at the CB1 receptor to inhibit adenylate cyclase activity via a pertussis toxin-sensitive G-protein. Within the striatum, CB1 receptors have been shown to be localized on the same neurons as Gi-coupled dopamine D2 receptors. In this study we have examined the interactions of CB1 and D2 receptors on adenylate cyclase. In striatal neurons in primary culture, both the CB1 receptor agonist [3-(1, 1-dimethylheptyl)-11-hydroxy-Delta8tetrahydrocannabinol] (HU210) and the D2 receptor agonist quinpirole inhibited forskolin-stimulated cAMP accumulation when applied separately. In contrast, HU210 and quinpirole in combination augmented cAMP accumulation. This augmentation was blocked by the CB1 receptor antagonist SR141716A or the D2 antagonist sulpride. Pertussis toxin treatment of striatal neurons prevented the inhibition of cAMP accumulation by D2 receptors but unmasked a cannabinoid receptor-mediated stimulatory effect on cAMP accumulation. The cannabinoid receptor-stimulated accumulation of cAMP was blocked in a concentration-dependent manner by SR141716A, suggesting that the response was regulated through the CB1 receptor. Similar augmentation of cAMP accumulation after pertussis toxin treatment was observed in Chinese hamster ovary (CHO) cells transfected with, and stably expressing, the CB1 receptor. This stimulation of cAMP was not Ca2+-sensitive and was unaffected by a range of protein kinase inhibitors. Treatment of the pertussis toxin-treated cells with cholera toxin before CB1 receptor activation amplified the stimulatory pathway, suggesting that this response was mediated through a Gs-type G-protein. Stimulation of cAMP accumulation was not observed after pertussis toxin treatment of CHO cells expressing the human CB2 receptor, suggesting that this novel signaling pathway is unique to the cannabinoid CB1 receptor.

Animals↗

PubMed1996

Isolation and measurement of the endogenous cannabinoid receptor agonist, anandamide, in brain and peripheral tissues of human and rat.

Anandamide (arachidonylethanolamide) is a novel lipid neurotransmitter first isolated from porcine brain which has been shown to be a functional agonist for the cannabinoid CB1 and CB2 receptors. Anandamide has never been isolated from human brain or peripheral tissues and its role in human physiology has not been examined. Anandamide was measured by LC/MS/MS and was found in human and rat hippocampus (and human parahippocampal cortex), striatum, and cerebellum, brain areas known to express high levels of CB1 cannabinoid receptors. Significant levels of anandamide were also found in the thalamus which expresses low levels of CB1 receptors. Anandamide was also found in human and rat spleen which expresses high levels of the CB2 cannabinoid receptor. Small amounts of anandamide were also detected in human heart and rat skin. Only trace quantities were detected in pooled human serum, plasma, and CSF. The distribution of anandamide in human brain and spleen supports its potential role as an endogenous agonist in central and peripheral tissues. The low levels found in serum, plasma, and CSF suggest that it is metabolized in tissues where it is synthesized, and that its action is probably not hormonal in nature.

Animals↗

PubMed1996

Identification and molecular characterization of a m5 muscarinic receptor in A2058 human melanoma cells. Coupling to inhibition of adenylyl cyclase and stimulation of phospholipase A2.

We report the identification and biochemical characterization of an endogenous m5 muscarinic acetylcholine receptor (mAChR) in the A2058 human melanoma cell line. This is the first demonstration of a m5AChR outside the central nervous system. The unusual effector coupling of this endogenous m5AChR is presented. The coding region amplified by polymerase chain reaction was identical to the known m5AChR sequence. Binding studies indicated a Kd of 99 +/- 6 pM and a Bmax of 45 +/- 4 fmol/mg membrane protein. This m5AChR coupled to stimulation of arachidonic acid release and to a 50% inhibition of forskolin-stimulated cAMP accumulation. The inhibition of cAMP production was insensitive to pertussis toxin treatment, but was dependent upon extracellular calcium. In contrast to the odd mAChR pattern, no cAMP was produced in response to carbachol (CC) stimulation. Moreover, no release of inositol phosphates could be measured after CC treatment despite the presence of at least 2 phospholipase C isoforms in A2058 cells. CC-stimulated arachidonic acid release (EC50 = 17.8 +/- 0.1 microM) was dependent upon external Ca2+, with marked reduction after coincubation with EGTA, Co2+, or high doses of verapamil (IC50 = 166 microM) or diltiazem (IC50 = 243 microM). Brief exposure to phorbol 12-myristate 13-acetate augmented CC-stimulated arachidonic acid release, whereas prolonged phorbol 12-myristate 13-acetate treatment resulted in down-regulation of release. Activation of the m5AChR resulted in Ca2+ influx that was attenuated by muscarinic antagonism and removal of extracellular Ca2+. A2058 cells exposed to CC had no alteration of cell shape or growth potential in monolayer culture, however, a statistically significant reduction in density-independent growth was observed over the range of CC concentrations from 0.1 to 100 microM. This endogenous m5AChR has a novel signal transduction coupling profile and receptor activation reduces clonogenic potential.

Adenylyl Cyclase Inhibitors↗

PubMed1996

The third intracellular domain of the m3 muscarinic receptor determines coupling to calcium influx in transfected Chinese hamster ovary cells.

The m2 and m3 muscarinic acetylcholine receptors were expressed in CHO cells and were shown to couple to the release of calcium from intracellular stores. The m3 receptor, but not the m2 receptor, also coupled to calcium influx. Chimeric m2/m3 receptors were used to determine the structural domain of the m3 receptor linked to the regulation of calcium influx. It was found that the third intracellular loop of m3 receptor plays a fundamental role in regulating Ca2+ influx predicted to occur through Ca2+ channels located in the plasma membrane in CHO cells.

Adenosine Triphosphate↗

PubMed1996

Independent induction of morphological transformation of CHO cells by receptor-activated cyclic AMP synthesis or by receptor-operated calcium influx.

Morphological transformation of Chinese hamster ovary (CHO) cells can be induced by exogenous addition of cyclic AMP (cAMP) or through the stimulation of G protein-coupled receptors ectopically expressed in these cells. The morphological transformation has been shown to represent a phenotypic suppression of CHO cell tumorigenic potential. Studies were undertaken to determine which receptor-activated signal transduction pathway initiates the progression from a tumorigenic to a non-tumorigenic phenotype. Stimulation of CHO cells expressing the dopamine D1 receptor (CHOD1) with a D1 selective agonist, SKF38393, resulted in an increase in cAMP accumulation which correlated with morphologic transformation. SKF38393 had no effect on intracellular calcium levels, arguing against a requirement for phospholipase C or calcium mobilization in the D1-stimulated morphology change. In contrast, stimulation of muscarinic m5 (CHOm5) or vasopressin V1a (CHOV1a) receptors expressed in CHO cells with carbachol or arginine vasopressin (AVP), respectively, did not result in an increase in intracellular calcium and a morphology change. The time course of carbachol-stimulated calcium influx correlated with the time course of morphological transformation, but not with carbachol-stimulated cAMP or inositol, 1,4,5-trisphosphate (IP3) accumulation. Furthermore, no increase in cAMP accumulation was observed in AVP-stimulated CHOV1a cells, suggesting a cAMP-independent stimulation of the transformation process. Carbachol-stimulated CHO cells expressing the m2 muscarinic receptor (CHOm2) failed to undergo a morphological transformation, yet released IP3. Therefore, phospholipase C-mediated signal transduction is not sufficient for the morphological transformation of CHO cells. It appears that receptor-stimulated morphologic transformation of CHO cells can be induced via two independent signaling pathways, mediated by adenylate cyclase or receptor-operated calcium channels.

2,3,4,5-Tetrahydro-7,8-dihydroxy-1-phenyl-1H-3-ben↗

PubMed1996

GABAA receptors modulate early spontaneous excitatory activity in differentiating P19 neurons.

P19 embryonic carcinoma (EC) stem cells are pluripotent and are efficiently induced to differentiate into neurons and glia with retinoic acid (RA) treatment. Within 5 days, a substantial number of differentiating P19 cells express gene products that are characteristic of a neuronal phenotype. P19 neurons were used as a model to explore the relationship between neuronal "differentiation" in vitro and the acquisition of gamma-aminobutyric acid (GABAA) receptors and functional GABA responses. Pulse-labeling experiments using bromodeoxyuridine indicated that all neurons had become postmitotic within 3-4 days after treatment with RA. This was confirmed by a reduction in the immunocytochemical detection of the undifferentiated stem cell antigen SSEA-1. Subsequently, a transient expression of nestin was observed during the first 5 days in vitro (DIV) after exposure to RA. By 5-10 DIV after RA, a significant number of neurons (approximately 80-90%) expressed immunocytochemically detectable glutamate decarboxylase and GABA coincident with the acquisition of membrane binding sites for tetanus toxin. These phenotypic markers were maintained for > 30 DIV after RA. Under current-clamp conditions, random, low-amplitude, spontaneous electrical activity appeared in neurons within the first few days after RA treatment and this was blocked by the specific GABAA receptor antagonist bicuculline. Thereafter, the appearance and progressive increases in the frequency of spontaneous action potentials in P19 neurons were observed that were similarly attenuated by bicuculline. In neurons > 5 DIV after RA, exogenous application of GABA elicited similar action potentials. The onset of excitatory responses to GABA or muscimol in voltage-clamped neurons appeared immediately after the cessation of neuronal mitosis and before the previously reported acquisition of responses to glutamate. In fura-2 imaging studies, the exogenous application of GABA resulted in neuron-specific increases in intracellular Ca2+. Thus, P19 neurons provide an in vitro model for the study of the early acquisition and properties of electrical excitability to GABA and the expression of functional GABAA receptors.

Action Potentials↗

PubMed1996

Muscarinic receptor activated Ca2+ channels in non-excitable cells.

We have provided preliminary characterization of a single channel Ca2+ conductance in CHO cells. We have demonstrated that the channel conducts Ca2+, is regulated by m5 receptors, is voltage-independent, has an extremely low conductance, and is second messenger-independent. This channel may be the receptor-operated channel required for downstream activation of several signaling events. It is not known what other cell types express the channel or if it is one of a larger group of related channels. It seems likely that Ca2+ influx-dependent signaling pathways, activated by the muscarinic m5 receptor, would utilize a plasma membrane resident Ca2+ channel to provide a steady source of Ca2+ from outside the cell. The transient nature of IP3-activated increases in intracellular Ca2+ make it an unlikely source of the sustained Ca2+ rise required for phospholipase regulation. This is especially surprising, since levels of intracellular Ca2+ achieved from the release of intracellular Ca2+ stores can be at least one order of magnitude higher than those achieved from extracellular influx (Berridge, 1993). The phospholipase A2 and phospholipase D involved in muscarinic receptor-mediated signaling have not been purified or cloned. It is possible that receptor-activated and Ca2+ influx-dependent phospholipases are integral membrane proteins located adjacent to both receptors and channels. The phospholipases may also translocate to the membrane following activation where they would gain access to the continuous Ca2+ flow. Purification and cloning of this and other related channels should provide better insight into their role in cell signaling.

Animals↗

PubMed1995

Extrapituitary expression of the rat V1b vasopressin receptor gene.

[Arg8]vasopressin (AVP) stimulates adrenocorticotropic hormone release from the anterior pituitary by acting on the V1b AVP receptor. This receptor can be distinguished from the vascular/hepatic V1a and renal V2 AVP receptors by its differential binding affinities for structural analogous of AVP. Recent studies have shown that the cloned V1a and V2 receptors are structurally related. We have isolated a clone encoding the V1b receptor from a rat pituitary cDNA library using polymerase chain reaction (PCR)-based methodology. The rat V1b receptor is a protein of 421 amino acids that has 37-50% identity with the V1a and V2 receptors. Homology is particularly high in the seven putative membrane-spanning domains of these guanine nucleotide-binding protein-coupled receptors. Expression of the recombinant receptor in mammalian cells shows the same binding specificity for AVP agonists and antagonists as the rat pituitary V1b receptor. AVP-stimulated phosphotidylinositol hydrolysis and intracellular Ca2+ mobilization in Chinese hamster ovary or COS-7 cells expressing the cloned receptor suggest second messenger signaling through phospholipase C. RNA blot analysis, reverse transcription PCR, and in situ hybridization studies reveal that V1b receptor mRNA is expressed in the majority of pituitary corticotropes as well as in multiple brain regions and a number of peripheral tissues, including kidney, thymus, heart, lung, spleen, uterus, and breast. Thus, the V1b receptor must mediate some of the diverse biological effects of AVP in the pituitary as well as other organs.

Amino Acid Sequence↗

PubMed1995

Vasopressin Vla receptor-stimulated phospholipase D: differential regulation of transphosphatidylation and phospholipid hydrolysis by protein kinase C [corrected].

Phospholipase D belongs to a group of membrane associated phospholipases which have been shown to be activated by G-protein coupled neurotransmitter receptors. Phosphatidylcholine is the primary substrate for phospholipase D generating phosphatidic acid (PA) and choline. In the presence of 1% ethanol, phospholipase D catalyzes a transphosphatidylation reaction generating phosphatidylethanol (PEt) which is an indicator of phospholipase D activation. In the present study, we utilized Chinese hamster ovary (CHO) cells stably transfected with and expressing a rat V1a vasopressin receptor to study the regulation of phospholipase D by protein kinase C and calcium. Arginine-vasopressin (AVP) stimulated the release of 3H-PEt and 3H-PA in cells pre-labelled overnight with 3H-palmitic acid. The phorbol ester, phorbol 12-myristate 13-acetate (PMA), stimulated the release of PEt and PA that was additive with AVP over 15 min. However, long-term stimulation with PMA, which desensitizes protein kinase C, decreased PEt production while simultaneously increasing PA production. Differential regulation of PEt and PA production by PMA suggests the existence of more than one phospholipase D isoenzyme. Though differentially regulated by protein kinase C, both AVP-stimulated PEt and PA production required extracellular and not intracellular calcium.

Animals↗

PubMed1995

Dopamine D1 receptor regulation of phospholipase C.

Dopamine is an endogenous catecholamine which exerts its actions by occupancy of specific receptors. Dopamine receptors are classified into two main groups: the two cloned D1-like receptors (D1A and D1B in rats; D1B is also known as D5 in humans) are linked to stimulation of adenylyl cyclase, while the three cloned D2-like receptors (D2 or D2A, D3 or D2B, D4 or D2C) are linked to inhibition of adenylyl cyclase. All these dopamine receptors originally cloned from the brain are expressed in tissues outside the central nervous system including the kidney. Dopamine regulates many cellular activities, including transmembrane ion transport. Activation of D1-like receptor decreases sodium transport by cAMP dependent and cAMP independent mechanisms. Dopamine, via D1-like receptors, may inhibit Na+/H+ exchange activity in renal brush border membranes by a cAMP independent/Gs alpha-linked mechanism. Another cAMP independent pathway of sodium transport inhibition is mediated by phospholipase C, which has several isoforms (PLC beta, PLC gamma, and PLC delta with several members in each). Catecholamines stimulate expression and activity of phospholipase C isoforms in a concentration, time, and receptor-dependent as well as regional and subcellular compartmental-specific manner. In renal cortical membranes, intrarenal administration of norepinephrine for 3-4 h increases PLC beta expression and activity but has no effect on PLC gamma activity. In contrast, intrarenal administration of a D1 agonist for 3-4 h increases PLC beta 1 but decreases PLC gamma expression and activity.(ABSTRACT TRUNCATED AT 250 WORDS)

Animals↗