The relationship of hormone-sensitive and hormone-insensitive phosphatidylinositol to phosphatidylinositol 4,5-bisphosphate in the WRK-1 cell

We have previously characterized two distinct pools of phosphatidylinositol (PI) in the WRK-1 rat mammary tumor cell, one whose metabolism is enhanced in response to vasopressin and another which is insensitive to hormonal manipulation. The purpose of the present study was to examine the relationship between cellular phosphatidylinositol 4,5-bisphosphate (PIP2) and each of the two PI pools. We have found that in WRK-1 cells, vasopressin induces the rapid loss of PIP2 and the accumulation of inositol phosphates. By making use of kinetic differences in 32Pi uptake into the two pools of PI and assessing radioactivity levels in the 1-phosphate of PIP2, we have determined that hormone-sensitive PI is the precursor of approximately 60% of the cellular PIP2; the remainder is synthesized from the hormone-insensitive pool. Additional data indicate that PIP2 derived from hormone-sensitive PI is likewise hormone-sensitive, while that synthesized from hormone-insensitive PI remains stable over a long period of time and is not affected by the presence of vasopressin.     

Characterization of the vasopressin receptor on WRK-1 cells

Specific vasopressin binding to WRK-1 rat mammary tumor cells was assessed and compared with vasopressin-induced alterations in phosphatidylinositol metabolism. Scatchard analysis revealed the presence of two binding sites: a saturable, high affinity site with a dissociation constant of 1 X 10(-9) M and an n of 2700 sites per cell, and a nonsaturable, apparent lower affinity site. The higher affinity site appeared to have V1a specificity and to correlate with vasopressin's ability to stimulate phosphatidylinositol turnover in the cells.     

Inositol metabolism in WRK-1 cells. Relationship of hormone-sensitive to -insensitive pools of phosphoinositides

Previous studies have indicated the existence of two separate pools of phosphoinositides in WRK-1 cells; one is labile and hormone-sensitive with respect to turnover, while the other is stable. Hormonal stimulation results in a rapid increase in 32Pi incorporation into the sensitive pool, while in the absence of hormone, incorporation of 32Pi into this pool is slow. Results are quite different when [3H]inositol is the precursor utilized. Incorporation of [3H]inositol into hormone-sensitive phosphoinositides is not stimulated in the presence of hormone, suggesting entry of this exogenous precursor into the cycle by a route other than the resynthetic phase of the cycle. Furthermore, failure of hormone to induce loss of [3H]phosphoinositide in pulse-chase experiments in the absence of lithium suggests reutilization of the [3H]inositol moiety generated by phosphodiesteratic cleavage of hormone-sensitive phosphoinositide. Time course studies indicate that the relative rates of incorporation of [3H]inositol into sensitive and insensitive phosphoinositide remain constant from 2 to 24 h. Several factors are capable of increasing [3H]inositol incorporation into hormone-insensitive phosphoinositide including vasopressin, calcium ionophores, and manganese. On the other hand, vasopressin treatment appears to decrease incorporation of [3H]inositol into the hormone-sensitive pool, probably by shifting the equilibrium between phosphoinositides and inositol phosphates, since the decrease in radioactivity observed in the phosphoinositides is equaled by the increase observed in that in the inositol phosphates.     

Evidence for a single pool of myo-inositol in hormone-responsive WRK-1 cells

Previous reports have suggested the existence of at least two pools of cellular myo-inositol (Ins); it has been further hypothesized that only one of these pools is utilized during hormone-activated, cyclic phosphatidylinositol (PtdIns) resynthesis. In an effort to investigate this possibility, we have undertaken kinetic studies of Ins metabolism in WRK-1 cells. Our results indicate that a single pool of Ins is involved in both basal and activated PtdIns synthesis. Ins generated by the hydrolysis of phosphatidylinositol 4,5-bisphosphate (PtdInsP₂) mixes with the existing pool of free Ins and is not used exclusively for resynthesis of PtdIns. © 1995 Wiley-Liss, Inc.     

Calcium and the phosphoinositide cycle in WRK-1 cells. Effects of A23187 on metabolism of specific phosphatidylinositol pools

WRK-1 cells possess a labile, hormone-sensitive pool of phosphatidylinositol which appears to be separate from the stable, hormone-insensitive phosphatidylinositol. It is the sensitive pool which turns over in response to treatment with vasopressin. Addition of the calcium ionophore A23187, on the other hand, selectively stimulates precursor incorporation into the hormone-insensitive pool of phosphatidylinositol, while causing nonspecific breakdown of both pools. The polyphosphoinositides are similarly affected. Ionophore-stimulated breakdown appears to be predominantly phospholipase C-mediated, since there is a concomitant increase in inositol phosphates. These inositol phosphates are localized predominantly in the extracellular medium. Permeabilization of the cells may explain the extracellular location of the breakdown products. When added together with the hormone, A23187, at concentrations greater than 5 X 10(-6) M, inhibits both hormone-induced synthesis and breakdown of phosphatidylinositol. Omission of calcium from the medium abolishes the effects of the ionophore.     

Effect of dual agonists on phosphoinositide pools in WRK-1 cells.

Both vasopressin and bradykinin activate the phosphoinositide cycle in WRK-1 rat mammary tumour cells. When the two agonists are added simultaneously, partial additivity is observed with respect to disappearance of prelabelled phosphoinositides and accumulation of inositol phosphates; no additivity is observed with respect to resynthesis of phosphatidylinositol as assessed by monitoring [32P]Pi incorporation. Lack of complete additivity can be explained, at least in part, by heterologous desensitization. In order to determine whether the two agonists were accessing a common or individual hormone-sensitive phosphoinositide pools, cells were incubated with [32P]Pi in the presence of either vasopressin or bradykinin and subsequently restimulated with the alternative agonist. The lipid pool labelled in the presence of either agonist was sensitive to subsequent treatment by the other ligand, suggesting a common phosphoinositide pool. However, when cells were incubated with [32P]Pi in the absence of agonists, the time course of labelling of the hormone-sensitive pool was different for bradykinin and vasopressin, with that for bradykinin becoming labelled within a much shorter time. Thus although there is a significant overlap between the phosphoinositide pools responding to vasopressin and bradykinin, there is a small fraction of the hormone-sensitive lipid which responds only to bradykinin.     

Phorbol ester inhibition of the hormone-stimulated phosphoinositide cycle in WRK-1 cells.

WRK-1 rat mammary tumour cells respond to vasopressin with increased accumulation of inositol phosphates as well as increased precursor incorporation into phosphatidylinositol. The phorbol ester, phorbol 13-myristate 12-acetate (PMA) inhibits by 80% both inositol phosphate accumulation and increased precursor incorporation. This inhibition is much less evident at early times (2 min) than at later times (25 min). The vasopressin-induced rise in cytosolic free Ca2+ is inhibited in a similar manner. Oleoylacetylglycerol is inactive with respect to inhibition of vasopressin-induced increases in incorporation of 32P into phosphoinositides. PMA has no effect on vasopressin binding at saturating concentrations of the hormone and does not affect the binding affinity.     

Maintenance of hormone-sensitive phosphoinositide pools in the plasma membrane requires phosphatidylinositol 4-kinase IIIalpha

Type III phosphatidylinositol (PtdIns) 4-kinases (PI4Ks) have been previously shown to support plasma membrane phosphoinositide synthesis during phospholipase C activation and Ca²⁺ signaling. Here, we use biochemical and imaging tools to monitor phosphoinositide changes in the plasma membrane in combination with pharmacological and genetic approaches to determine which of the type III PI4Ks (α or β) is responsible for supplying phosphoinositides during agonist-induced Ca²⁺ signaling. Using inhibitors that discriminate between the α- and β-isoforms of type III PI4Ks, PI4KIIIα was found indispensable for the production of phosphatidylinositol 4-phosphate (PtdIns4P), phosphatidylinositol 4,5-bisphosphate [PtdIns(4,5)P₂], and Ca²⁺ signaling in angiotensin II (AngII)-stimulated cells. Down-regulation of either the type II or type III PI4K enzymes by small interfering RNA (siRNA) had small but significant effects on basal PtdIns4P and PtdIns(4,5)P₂ levels in ³²P-labeled cells, but only PI4KIIIα down-regulation caused a slight impairment of PtdIns4P and PtdIns(4,5)P₂ resynthesis in AngII-stimulated cells. None of the PI4K siRNA treatments had a measurable effect on AngII-induced Ca²⁺ signaling. These results indicate that a small fraction of the cellular PI4K activity is sufficient to maintain plasma membrane phosphoinositide pools, and they demonstrate the value of the pharmacological approach in revealing the pivotal role of PI4KIIIα enzyme in maintaining plasma membrane phosphoinositides.     

Characterization of hormone-sensitive adenylate cyclase in rabbit gallbladder mucosa

1. We have developed a plasma membrane preparation from the mucosal epithelium of rabbit gallbladder and have characterized the hormonal sensitivity of adenylate cyclase in this preparation. 2. Basal activity is low and is stimulated by GTP and GppNHp. Hormonal stimulation is largely dependent on exogenous guanine nucleotide. 3. Several prostaglandins (E1 approximately E2 greater than A1 greater than B1), vasoactive intestinal peptide and the beta-adrenergic agonist, isoproterenol, stimulate mucosal adenylate cyclase activity; a variety of peptides and neurotransmitters (secretin, cholecystokinin, arg-vasopressin, oxytocin, histamine, dopamine and serotonin) are without effect. 4. The data support the hypothesis that the inhibitory effect of prostaglandins, vasoactive intestinal peptide, and isoproterenol on gallbladder fluid absorption in certain species may be mediated by cyclic AMP. 5. The membrane preparation should be useful in further characterizing hormone receptor-transducer interactions of the gallbladder mucosal epithelium.     

Mobilization of the hormone-sensitive calcium pool increases hepatocyte tight junctional permeability in the perfused rat liver

Hepatocyte tight junctional permeability has been shown to be regulated by hormones that exert their effects via phospholipase C activation. However, the precise transduction pathway involved in this effect is not known. The present study has employed the selective inhibitor of microsomal Ca2+ sequestration, 2,5-di(tert-butyl)-1,4-benzohydroquinone (tBuBHQ), to examine the effect of the mobilization of the endoplasmic reticular Ca2+ pool on tight junctional permeability in the perfused rat liver. Infusion of tBuBHQ followed by a bolus infusion of horseradish peroxidase (HRP) resulted in a significant increase in the first peak of biliary HRP, a measure of junctional permeability, whereas transcellular (vesicular) transport of HRP was not affected. Therefore, we conclude that the effect of hormones on tight junctional permeability is mediated, at least in part, by the mobilization of intracellular Ca2+.     

Extensive incorporation of dietary delta-5,11,14 eicosatrienoate into the phosphatidylinositol pool

The acyl composition of phosphatidylinositol (PI) is remarkably resistant to dietary fatty acid modification. To investigate the basis of this selectivity, we have probed fatty acids lacking the usual methylene interrupted double bonds. When mice were fed delta-5,11,14 20:3 as 3% of total lipid, this fatty acid, lacking the delta-8 double bond essential for eicosanoid synthesis, replaced a significant quantity of 20:4 (n-6) in PI, but not PC and PE. By altering the acyl composition of PI, novel second messengers may be formed. This fatty acid structure thus provides a unique nutritional tool for investigating the basis of PI acyl specificity, and for determining the metabolic consequences of acyl alteration, in vivo.     

Lipolytic stimulation modulates the subcellular distribution of hormone-sensitive lipase in 3T3-L1 cells

3T3-L1 cells have been a useful model system for studying adipocyte differentiation and metabolism. They acquire a hormone-sensitive lipase during differentiation (Kawamura, M., et al. 1981. Proc. Natl. Acad. Sci. USA. 78: 732-735). In the present study the control of lipolysis in these cells was investigated. Basal glycerol release from cell monolayers was 437 nmol/mg protein per hr, and could be stimulated approximately 6-fold by exposure to 1 microM isoproterenol. Subcellular fractionation of stimulated cells revealed a redistribution of triglyceride lipase activity: loss from the infranatant fraction and increase in the pellet fraction. The redistribution was dosage-dependent and reversible. Treatment of intact cells with 8-bromoadenosine 3':5' cyclic monophosphate elicited similar redistribution of the lipase activity; however, disruption and incubation of untreated cells in the presence of ATP and either cyclic AMP or the catalytic subunit from cAMP-dependent protein kinase did not. The lipase activity in the pellet fraction was increased 3- to 4-fold after maximal lipolytic stimulation of intact cells, whereas phosphorylation of the enzyme in vitro yielded 1.4- to 1.6-fold stimulation in all subcellular fractions from untreated cells. The lipase found in the particulate fraction has the same properties as the previously characterized infranatant enzyme. It is suggested that interaction of the lipase with substrate and associated intracellular membranes may be a novel feature of the regulation of lipolysis.     

Perilipin targets a novel pool of lipid droplets for lipolytic attack by hormone-sensitive lipase

Adipocytes serve as the principal energy reservoir of the body; however, the subcellular organization of the machinery regulating lipid trafficking and metabolism is poorly understood. Mobilization of stored triglyceride is thought be controlled by interactions among intracellular lipases and proteins that coat lipid storage droplets. A major limitation of previous studies of hormone-mediated lipolysis, however, is the use of cultured model adipocytes whose three-dimensional architectures do not resemble those in real adipose tissue. To address this limitation, we investigated the intracellular targeting of perilipin, a major lipid coat protein, and hormone-sensitive lipase in three preparations that exhibit more appropriate morphologies: 3T3-L1 adipocytes grown in three-dimensional matrix, dissociated mature adipocytes from mouse adipose tissue, and adipocytes within intact fat pads. High resolution imaging of native and fluorescently tagged proteins indicate that: 1) perilipin preferentially targets a special class of peripheral lipid storage droplets, but not the major or central lipid storage droplets, 2) the peripheral droplets are the sites of attack by hormone-sensitive lipase, and 3) perilipin and hormone-sensitive lipase are continuously colocalized following lipolytic activation. These results indicate that in white adipose tissue, lipolysis takes place in a specialized subcellular domain that is distinct from the major lipid storage site and is defined by perilipin.     

Membrane phospholipids and hormone-sensitive lipolysis in fat cells

Isolated fat cells from rats which have been made hypothyroid do not give a lipolytic response to catecholamines. A recent report has suggested that catecholamine-sensitive lipolysis may be correlated with an “unmasking” of receptors by linoleic acid rich phospholipids in the fat cell membrane. No apparent differences in phospholipid fatty acid composition could be found in membrane “ghosts” prepared from normal and hypothyroid rats.     

Sac1 lipid phosphatase and Stt4 phosphatidylinositol 4-kinase regulate a pool of phosphatidylinositol 4-phosphate that functions in the control of the actin cytoskeleton and vacuole morphology

Synthesis and turnover of phosphoinositides are tightly regulated processes mediated by a set of recently identified kinases and phosphatases. We analyzed the primary role of the phosphoinositide phosphatase Sac1p in Saccharomyces cerevisiae with the use of a temperature-sensitive allele of this gene. Our analysis demonstrates that inactivation of Sac1p leads to a specific increase in the cellular levels of phosphatidylinositol 4-phosphate (PtdIns(4)P), accompanied by changes in vacuole morphology and an accumulation of lipid droplets. We have found that the majority of Sac1p localizes to the endoplasmic reticulum, and this localization is crucial for the efficient turnover of PtdIns(4)P. By generating double mutant strains harboring the sac1(ts) allele and one of two temperature-sensitive PtdIns 4-kinase genes, stt4(ts) or pik1(ts), we have demonstrated that the bulk of PtdIns(4)P that accumulates in sac1 mutant cells is generated by the Stt4 PtdIns 4-kinase, and not Pik1p. Consistent with these findings, inactivation of Sac1p partially rescued defects associated with stt4(ts) but not pik1(ts) mutant cells. To analyze potential overlapping functions between Sac1p and other homologous phosphoinositide phosphatases, sac1(ts) mutant cells lacking various other synaptojanin-like phosphatases were generated. These double and triple mutants exacerbated the accumulation of intracellular phosphoinositides and caused defects in Golgi function. Together, our results demonstrate that Sac1p primarily turns over Stt4p-generated PtdIns(4)P and that the membrane localization of Sac1p is important for its function in vivo. Regulation of this PtdIns(4)P pool appears to be crucial for the maintenance of vacuole morphology, regulation of lipid storage, Golgi function, and actin cytoskeleton organization.     

Characterization of neurotransmitter receptor-mediated phosphatidylinositol hydrolysis in the rat hippocampus

The stimulation of phosphatidylinositol hydrolysis by various neurotransmitter agonists was investigated in rat hippocampal slices using a rapid and sensitive radioisotopic method. Slices were preincubated with [3H]-myo-inositol and the accumulation of [3H]-myo-inositol-1-phosphate induced by various agonists was determined in the presence of 10 mM lithium. The latter resulted in a marked amplification of the response to all agonists tested. The agonist-induced accumulation of [3H]-myo-inositol-1-phosphate was dependent on tissue, lithium, [3H]-myo-inositol concentration, as well as incubation time. The hydrolysis of phosphatidylinositol in hippocampal slices is induced by carbachol, serotonin, norepinephrine and phenylephrine. The carbachol-induced response is sensitive to atropine, a muscarinic-cholinergic antagonist, but not mecamylamine a nicotinic-cholinergic antagonist, while that of norepinephrine is blocked by the alpha 1 adrenoreceptor antagonist prazosin, but not the specific alpha 2 antagonist Rx 781094. Phenylephrine, another alpha 1 adrenoreceptor agonist produced a partial or submaximal response when compared to norepinephrine. The concentration response curve for serotonin-induced phosphatidylinositol hydrolysis is bimodal and the effect is blocked by metergoline, but not mianserin, indicating that the effect of serotonin in the hippocampus may be mediated by 5HT1 receptors. Our results suggest that the measurement of agonist-induced [3H]-myo-inositol-1-phosphate accumulation, in the presence of lithium, represents a sensitive method for studying a number of receptor-mediated events in brain.     

Characterization of a novel testicular form of human hormone-sensitive lipase

Hormone-sensitive lipase (HSL) is an esterase and lipase, which are essential for spermatogenesis. Two HSL mRNAs are expressed in human testis. A long form is encoded by a testis-specific exon and nine exons common to testis and adipocyte HSL. Here we show that the short-form 3.3-kb mRNA possesses a unique 5' end that is transcribed from a novel testis-specific exon. The corresponding protein is similar to the 775-amino-acid-long adipocyte HSL. Immunohistochemistry experiments on human testis sections revealed that the long form is strictly expressed in haploid germ cells whereas the short form is expressed in interstitial and tubular somatic cells as well as premeiotic germ cells.     

Activation of the phosphatidylinositol cycle in spreading cells

Metabolites of the phosphatidylinositol cycle were analyzed in BHK-21 (C13) cells spreading on fibronectin-coated culture plates in comparison with attached nonspreading cells 45 min after plating. Among the water-soluble metabolites (glycerophosphoinositol, inositol, inositol monophosphate, inositol bisphosphate, inositol trisphosphate, and inositol tetrakisphosphate), significant elevations were found for inositol monophosphate, inositol bisphosphate, and inositol tetrakisphosphate. In the lipid fraction, phosphatidylinositol 4-monophosphate and phosphatidylinositol 4,5-bisphosphate were significantly elevated. The activation of the phosphatidylinositol cycle in spreading versus nonspreading attached BHK-21 (C13) cells may be involved in the permissive effect of the extracellular matrix on cell proliferation.     

Mechanism of UV-induced deoxynucleoside triphosphate pool imbalance in CHO-K1 cells

Several laboratories have reported that exposure of cells to UV radiation results in a significant imbalance in deoxynucleoside triphosphate pool concentrations. In our CHO-K1 cells, a rapid drop in dCTP is accompanied by a rapid increase in dTTP. Examination of enzyme activities associated with synthesis/degradation of these molecules suggests that UV transiently enhances a putative dCTPase, dCMP deaminase and CdR kinase activities. This results in accumulation of excess dUMP which is probably converted to dTMP, then to dTTP. The absence of dCMP deaminase in V79 cells prohibits this rapid response in those cells. Moreover, significantly different dCMP deaminase activities were observed in CHO-K1 cells obtained from other laboratories, suggesting they, too, may respond differently to irradiation.     

Characterization of the functional interaction of adipocyte lipid-binding protein with hormone-sensitive lipase.

Hormone-sensitive lipase (HSL) is an intracellular lipase that plays an important role in the hydrolysis of triacylglycerol in adipose tissue. HSL has been shown to interact with adipocyte lipid-binding protein (ALBP), a member of the family of intracellular lipid-binding proteins that bind fatty acids and other hydrophobic ligands. The current studies have addressed the functional significance of the association and mapped the site of interaction between HSL and ALBP. Incubation of homogeneous ALBP with purified, recombinant HSL in vitro resulted in a 2-fold increase in substrate hydrolysis. Moreover, the ability of oleate to inhibit HSL hydrolytic activity was attenuated by co-incubation with ALBP. Co-transfection of Chinese hamster ovary cells with HSL and ALBP resulted in greater hydrolytic activity than transfection of cells with HSL and vector alone. Deletional mutations of HSL localized the region of HSL that interacts with ALBP to amino acids 192-200, and site-directed mutagenesis of individual amino acids in this region identified His-194 and Glu-199 as critical for mediating the interaction of HSL with ALBP. Interestingly, HSL mutants H194L and E199A, each of which retained normal basal hydrolytic activity, failed to display an increase in hydrolytic activity when co-transfected with wild type ALBP. Therefore, ALBP increases the hydrolytic activity of HSL through its ability to bind and sequester fatty acids and via specific protein-protein interaction. Thus, HSL and ALBP constitute a functionally important lipolytic complex.