Phospholipase C Isoforms Research Articles

Phospholipase C isoforms in mammalian spermatozoa: potential components of the sperm factor that causes Ca2+ release in eggs

Injection of a soluble protein factor from mammalian spermatozoa triggers Ca2+ oscillations in mammalian eggs similar to those seen at fertilization. This sperm factor also generates inositol 1,4,5-trisphosphate and causes Ca2+ release in sea urchin egg homogenates and frog eggs. Recent studies have indicated that the sperm factor may be an inositol-specific phospholipase C (PLC) activity. This study investigated whether any of the commonly known PLC isoforms are components of the sperm factor. PLCbeta, PLCgamma and PLCdelta isoforms were shown to be present in boar sperm extracts. However, upon column fractionation of sperm extracts, none of the PLC isoforms detected correlated with the ability to cause Ca2+ release in eggs. In addition to our previous work on recombinant PLCs, it was also shown that PLCdelta3, PLCdelta4 and its splice variant PLCdelta4 Alt1 fail to cause Ca2+ release. The recently discovered 255 kDa PLCepsilon isoform also appears unlikely to be a component of the sperm factor, as fractionation of sperm extracts on a gel filtration column demonstrated that the peak of Ca2+-releasing activity was associated with fractions of 30-70 kDa. These findings indicate that the sperm factor that triggers Ca2+ release in eggs does not appear to have a known PLC isoform as one of its components.

Phospholipase C isoforms in mammalian spermatozoa: potential components of the sperm factor that causes Ca2+ release in eggs.

Injection of a soluble protein factor from mammalian spermatozoa triggers Ca2+ oscillations in mammalian eggs similar to those seen at fertilization. This sperm factor also generates inositol 1,4,5-trisphosphate and causes Ca2+ release in sea urchin egg homogenates and frog eggs. Recent studies have indicated that the sperm factor may be an inositol-specific phospholipase C (PLC) activity. This study investigated whether any of the commonly known PLC isoforms are components of the sperm factor. PLCbeta, PLCgamma and PLCdelta isoforms were shown to be present in boar sperm extracts. However, upon column fractionation of sperm extracts, none of the PLC isoforms detected correlated with the ability to cause Ca2+ release in eggs. In addition to our previous work on recombinant PLCs, it was also shown that PLCdelta3, PLCdelta4 and its splice variant PLCdelta4 Alt1 fail to cause Ca2+ release. The recently discovered 255 kDa PLCepsilon isoform also appears unlikely to be a component of the sperm factor, as fractionation of sperm extracts on a gel filtration column demonstrated that the peak of Ca2+-releasing activity was associated with fractions of 30-70 kDa. These findings indicate that the sperm factor that triggers Ca2+ release in eggs does not appear to have a known PLC isoform as one of its components.

Gαq/11 Coupled to Mammalian Phospholipase C β3-like Enzyme Mediates the Ginsenoside Effect on Ca2+-activated Cl− Current in theXenopus Oocyte

Recently we demonstrated that ginsenosides, the active ingredients of Panax ginseng, enhanced Ca(2+)-activated Cl(-) current in the Xenopus oocyte through a signal transduction mechanism involving the activation of pertussis toxin-insensitive G protein and phospholipase C (PLC). However, it has not yet been determined precisely which G protein subunit(s) and which PLC isoform(s) participate in the ginsenoside signaling. To provide answers to these questions, we investigated the changes in ginsenoside effect on the Cl(-) current after intraoocyte injections of the cRNAs coding various G protein subunits, a regulator of G protein signaling (RGS2), and G beta gamma-binding proteins. In addition, we examined which of mammalian PLC beta 1-3 antibodies injected into the oocyte inhibited the action of ginsenosides on the Cl(-) current. Injection of G alpha(q) or G alpha(11) cRNA increased the basal Cl(-) current recorded 48 h after, and it further prevented ginsenosides from enhancing the Cl(-) current, whereas G alpha(i2) and G alpha(oA) cRNA injection had no significant effect. The changes following G alpha(q) cRNA injection were prevented when G beta(1)gamma(2) and G alpha(q) subunits were co-expressed by simultaneous injection of the cRNAs coding these subunits. Injection of cRNA coding G alpha(q)Q209L, a constitutively active mutant that does not bind to G beta gamma, produced effects similar to those of G alpha(q) cRNA injection. The effects of G alpha(q)Q209L cRNA injection, however, were not prevented by co-injection of G beta(1)gamma(2) cRNA. Injection of the cRNA coding RGS2, which interacts most selectively with G alpha(q/11) among various identified RGS isoforms and stimulates the hydrolysis of GTP to GDP in active GTP-bound G alpha subunit, resulted in a severe attenuation of ginsenoside effect on the Cl(-) current. Finally, antibodies against PLC beta 3, but not -beta 1 and -beta 2, markedly attenuated the ginsenoside effect examined at 3-h postinjection. These results suggest that G alpha(q/11) coupled to mammalian PLC beta 3-like enzyme mediates ginsenoside effect on Ca(2+)-activated Cl(-) current in the Xenopus oocyte.

Phospholipase Cβ4 and Protein Kinase Cα and/or Protein Kinase CβI Are Involved in the Induction of Long Term Depression in Cerebellar Purkinje Cells

Activation of the type-1 metabotropic glutamate receptor (mGluR1) signaling pathway in the cerebellum involves activation of phospholipase C (PLC) and protein kinase C (PKC) for the induction of cerebellar long term depression (LTD). The PLC and PKC isoforms that are involved in LTD remain unclear, however. One previous study found no change in LTD in PKCgamma-deficient mice, thus, in the present study, we examined cerebellar LTD in PLCbeta4-deficient mice. Immunohistochemical and Western blot analyses of cerebellum from wild-type mice revealed that PLCbeta1 was expressed weakly and uniformly, PLCbeta2 was not detected, PLCbeta3 was expressed predominantly in caudal cerebellum (lobes 7-10), and PLCbeta4 was expressed uniformly throughout. In PLCbeta4-deficient mice, expression of total PLCbeta, the mGluR1-mediated Ca(2+) response, and LTD induction were greatly reduced in rostral cerebellum (lobes 1-6). Furthermore, we used immunohistochemistry to localize PKCalpha, -betaI, -betaII, and -gamma in mouse cerebellar Purkinje cells during LTD induction. Both PKCalpha and PKCbetaI were found to be translocated to the plasmamembrane under these conditions. Taken together, these results suggest that mGluR1-mediated activation of PLCbeta4 in rostral cerebellar Purkinje cells induced LTD via PKCalpha and/or PKCbetaI.

Phospholipase C δ2 Expression Characterizes the Neoplastic Transformation of the Human Gastric Mucosa

The expression, cellular distribution, and activity of PIP(2)-specific phospholipase C (PLC) in healthy human gastric-mucosa cells have been recently studied in our laboratories and a direct evidence for an almost exclusive expression of PLC beta isoforms, with the exception of PLC beta4, has been provided. These results addressed our attention to possible modification of PLC expression and activity during neoplastic transformation of the human gastric mucosa. In the present article we present results indicating that PLC delta2 is markedly expressed in type II intestinal metaplasia and in the adenocarcinoma whereas traces of other PLC isoforms were sometime detected. Interestingly, we found that type I intestinal metaplasia was in the majority of the cases PLC delta2-negative, but when expressed, this type of metaplasia generally considered as benignant, always evolved toward neoplastic transformation. These results therefore readdress the question of surveillance of the patients with type I intestinal metaplasia and suggest that PLC delta2 expression might be a possible marker of gastric malignant transformation.

PTH stimulates PLCβ and PLCγ isoenzymes in rat enterocytes: influence of ageing

We previously reported that in rat duodenal cells (enterocytes), parathyroid hormone (PTH [1–34]: PTH) stimulates the hydrolysis of polyphosphoinositides by phospholipase C (PLC), generating the second messengers inositol trisphosphate (IP 3) and diacylglycerol (DAG) and that this mechanism is severely altered in old animals. In the present study, we show that PTH [1–34]-dependent IP 3 release in young rats was blocked to a great extent by an antibody against guanine nucleotide binding protein Gαq/11, indicating that the hormone activates a β isoform of PLC coupled to the α subunit of Gq/11. In addition, PTH rapidly (within 30 s, with maximal effects at 1 min) stimulated tyrosine phosphorylation of PLCγ in a dose-dependent fashion (10 −10–10 −7 M). The hormone response was specific as PTH [7–34] was without effects. The tyrosine kinase inhibitors, genistein (100 μM) and herbimycin (2 μM), suppressed PTH-dependent PLCγ tyrosine phosphorylation. Stimulation of PLCγ tyrosine phosphorylation by PTH [1–34] greatly decreased with ageing. PP1 (10 μM), a specific inhibitor of the Src family of tyrosine kinases, completely abolished PLCγ phosphorylation. The hormone-induced Src tyrosine dephosphorylation, a major mechanism of Src activation, an effect that was blunted in old animals. These results indicate that in rat enterocytes PTH generates IP 3 mainly through G-protein-coupled PLCβ and stimulates PLCγ phosphorylation via the nonreceptor tyrosine kinase Src. Impairment of PTH activation of both PLC isoforms upon ageing may result in abnormal hormone regulation of cell Ca 2+ and proliferation in the duodenum.

Ras Targets Phospholipase C

The family of phospholipase C (PLC) isoforms can be divided into three classes β, γ, and δ. Song et al. have identified a fourth class of PLC, PLCε, which has sequence similarity to the Caenorhabditis elegans PLC210, including two putative Ras-interacting domains (CDC25-like domain and an RA domain). However, it lacks the conserved pleckstrin homology domain and EF-hand domains of all other classes of PLC. Analysis of recombinant PLCε showed that the protein has phospholipase C activity with a dependence on calcium similar to that of other PLC isoforms. PLCε interacted with the guanosine triphosphatases Ras and Rap1, and this interaction led to the recruitment of PLCε to the plasma membrane or the perinuclear region, respectively, in cells transfected with a green fluorescent protein (GFP)-tagged PLCε and activated forms of Ras or Rap1. Furthermore, the translocation of PLCε to membranes was stimulated in cells treated with epidermal growth factor, and this effect was blocked by expression of dominant negative Ras, supporting the model that Ras recruits the PLCε to the membrane in response to external stimuli. C. Song, C.-D. Hu, M. Misago, K.-i. Kariya, Y. Yamawaki-Kataoka, M. Shibatohge, D. Wu, T. Satoh, T. Katoaka, Regulation of a novel human phospholipase C, PLCε, through membrane targeting by Ras. J. Biol. Chem. 276 , 2752-2757 (2001). [Abstract] [Full Text]

Pancreastatin, a chromogranin A-derived peptide, activates Gα 16 and phospholipase C-β 2 by interacting with specific receptors in rat heart membranes

Pancreastatin (PST) is one of the chromogranin A (CGA)-derived peptides with known biological activity. It has a general inhibitory effect on secretion in many exocrine and endocrine systems including the heart atrium. Besides, a role of PST as a counter-regulatory peptide of insulin action has been proposed in the light of its effects on glucose and lipid metabolism in the liver and adipose tissue, where receptors and signaling have been described. Gα q/11 pathway seems to mediate PST action. Since PST has been shown to function as a typical calcium-dependent hormone, and increased plasma levels have been found in essential hypertension correlating with catecholamines, we sought to study its possible interaction and signaling in heart membranes. Here, we are characterizing specific PST binding sites and signaling in rat heart membranes. We have found that PST receptor has a K d of 0.5 nM and a B max of 34 fmol/mg of protein. The PST binding is inhibited by guanine nucleotides, suggesting the functional coupling of the receptor with GTP binding proteins (G proteins). Moreover, PST dose-dependently increases GTP binding to rat heart membranes. Finally, we have studied PST signaling-effector system by measuring phospholipase C (PLC) activity using blocking antibodies against different G proteins and PLC isoforms. We have found that PST stimulates PLCβ 2>PLCβ 1>PLCβ 3 by activating Gα 16 in rat heart membranes. These data suggest that PST may modulate the cardiac function.

Aging and calcitriol regulation of IP3 production in rat skeletal muscle and intestine.

We previously reported that calcitriol [1,25(OH)2-vitamin D3] in rat skeletal muscle and duodenum stimulates the hydrolysis of polyphosphoinositides by phospholipase C (PLC), generating the second messengers inositol trisphosphate (IP3) and diacylglycerol (DAG), and that this mechanism is altered in old animals. As previously reported in muscle, we show in the present study that GTPgammaS (100 microM, 15 s), the non-hydrolyzable analogue of GTP, increased IP3 release from young rats duodenum to the same extent as 1 nM calcitriol (+ 100%), while GDPbetaS (100 microM) suppressed hormone-dependent IP3 production. Similarly to calcitriol, GTPgammaS response was diminished in old rats. Contrary to muscle, pretreatment with Bordetella pertussis toxin did not modify calcitriol-dependent IP3 in duodenum. The antibody, anti-G alpha q/11 (1:200) and anti-G alpha i (1:200) blocked calcitriol-dependent IP3 release in muscle from young rats, indicating that the hormone activates an isoform of PLC coupled to the alpha subunit of Gq/11 and possibly the betagamma subunits of Gi. The aged muscle was insensitive to anti G alpha i. In rat duodenum the hormone effects were suppressed by anti-Gq/11 both in young and aged animals. In 24-month-old rats, Gq/11 and Gi protein levels were greatly reduced both in muscle and duodenum, suggesting that a deficiency in G protein expression with aging may have important consequences for correct receptor/effector coupling and could explain age-related declines in the function of second messenger systems linked to G-proteins.

GTP Binding and Signaling by Gh/Transglutaminase II Involves Distinct Residues in a Unique GTP-binding Pocket

G(h) is a dual function protein. It has receptor signaling activity that requires GTP binding and Ca(2+)-activated transglutaminase (TGase) activity that is inhibited by GTP binding. G(h) shows no homology with other GTP-binding proteins, and its GTP-binding site has not been defined. Based on sequence analysis of [alpha-(32)P]GTP-photolabeled and proteolytically released internal peptide fragments, we report localization of GTP binding to a 15-residue segment ((159)YVLTQQGFIYQGSVK(173)) of the G(h) core domain. This was confirmed by site-directed mutagenesis; a G(h)/fXIIIA chimera (in which residues 162-179 of G(h) were substituted with the equivalent but nonhomologous region of the non-GTP-binding TGase factor XIIIA) and a G(h) point mutant, S171E, retained TGase activity but failed to bind and hydrolyze GTP and did not support alpha(1B)-adrenergic receptor signaling. Slight impairment of GTP binding (1.5-fold) and hydrolysis (10-fold) in the absence of altered TGase activity did not affect signaling by the mutant K173N. However, greater impairment of GTP binding (6-fold) and hydrolysis (50-fold) abolished signaling by the mutant K173L. Mutant S171C exhibited enhanced GTP binding and signaling. Thus, residues Ser(171) and Lys(173) are critical for both GTP binding and signaling but not TGase activity. Mutagenesis of residues N-terminal to Gly(170) impaired both GTP binding and TGase activity. From computer modeling of G(h), it is evident that the GTP-binding region identified here is distinct from, but interacts with, the TGase active site. Together with structural considerations of G(h) versus other GTP-binding proteins, these findings indicate that G(h) has a unique GTP-binding pocket and provide for the first time a mechanism for GTP-mediated regulation of the TGase activity of G(h).

Membrane signaling and progesterone in female and male osteoblasts. II. Direct involvement of G alpha q/11 coupled to PLC-beta 1 and PLC-beta 3.

We have shown that progesterone (10 pM-10 nM) and progesterone covalently bound to bovine serum albumin (P-CMO BSA; 100 pM-1 microM) rapidly increased (within 5 s) the cytosolic free Ca(2+) concentration and inositol 1,4,5 trisphosphate (InsP(3)) formation in confluent female and male rat osteoblasts via a pertussis toxin-insensitive G-protein. The activation of G-proteins coupled to effectors such as phospholipase C (PLC) is an early event in the signal transduction pathway leading to InsP(3) formation. We used antibodies against the various PLC isoforms to show that only PLC-beta1 and PLC-beta 3 were involved in the Ca(2+) mobilization and InsP(3) formation induced by both progestins in female and male osteoblasts, whereas PLC-beta 2, PLC-gamma 1, and PLC-gamma 2 were not. We also used antibodies against the subunits of heterotrimeric G-proteins to show that the activation of PLC-beta 1 and PLC-beta 3 by both progestins involved the G alpha q/11 subunit, which was insensitive to pertussis toxin, whereas G alpha i, G alpha s, and G beta gamma subunits were not. The membrane effects were independent of the concentration of nuclear progesterone receptor, because the concentration of nuclear progesterone receptors was lower in male than in female osteoblasts. These data suggest that progesterone and P-CMO BSA, which does not enter the cell, directly activate G-protein leading to the very rapid formation of second messengers without involving the nuclear receptor.

Concomitant activation of G i protein-coupled receptor and protein kinase C or phospholipase C is required for platelet aggregation

It has recently been suggested that the concomitant activation of two distinct G protein-coupled receptors (G i and G q) is essential for platelet aggregation: in fact, the thromboxane A 2 synthetic agonist, U46619, which causes the selective activation of G q, is not able to elicit fibrinogen receptor exposure unless ADP or epinephrine is present. In the present study we demonstrate that a direct G q activation is not required for platelet aggregation and that the activation of an enzyme downstream of G q, such as phospholipase C (PLC) or protein-kinase C (PKC), is sufficient for such a process. In fact, platelet aggregation occurred in response to the snake venom toxin convulxin, which activates the PLC isoform PLCγ2 or to cytosolic PKC activator phorbol 12-myristate 13-acetate (PMA) provided a G i protein-coupled receptor was activated by ADP or epinephrine. The evidence that the PKC inhibitor, Ro 31-8220 did not suppress platelet aggregation in response to convulxin plus ADP or epinephrine led us to conclude that PLC and PKC are both involved in platelet aggregation, although not concomitantly, provided a G i protein-coupled receptor is activated.

The Role of Individual SH2 Domains in Mediating Association of Phospholipase C-γ1 with the Activated EGF Receptor

The two SH2 (Src homology domain 2) domains present in phospholipase C-gamma1 (PLC-gamma1) were assayed for their capacities to recognize the five autophosphorylation sites in the epidermal growth factor receptor. Plasmon resonance and immunological techniques were employed to measure interactions between SH2 fusion proteins and phosphotyrosine-containing peptides. The N-SH2 domain recognized peptides in the order of pY1173 > pY992 > pY1068 > pY1148 >> pY1086, while the C-SH2 domain recognized peptides in the order of pY992 > pY1068 > pY1148 >> pY1086 and pY1173. The major autophosphorylation site, pY1173, was recognized only by the N-SH2 domain. Contributions of the N-SH2 and C-SH2 domains to the association of the intact PLC-gamma1 molecule with the activated epidermal growth factor (EGF) receptor were assessed in vivo. Loss of function mutants of each SH2 domain were produced in a full-length epitope-tagged PLC-gamma1. After expression of the mutants, cells were treated with EGF and association of exogenous PLC-gamma1 with EGF receptors was measured. In this context the N-SH2 is the primary contributor to PLC-gamma1 association with the EGF receptor. The combined results suggest an association mechanism involving the N-SH2 domain and the pY1173 autophosphorylation site as a primary event and the C-SH2 domain and the pY992 autophosphorylation site as a secondary event.

Cloning and characterization of a gene encoding phosphatidyl inositol-specific phospholipase C from Trypanosoma cruzi

A gene encoding phosphatidyl inositol-4,5-bisphosphate phospholipase C (PLC) was cloned from the protozoan parasite Trypanosoma cruzi. A partial cDNA encoding putative PLC was obtained by a polymerase chain reaction (PCR) using degenerate oligonucleotide primers corresponding to conserved regions of PLCs. A 2178-bp protein coding region of the T. cruzi PLC gene, composed from cDNA and genomic clones, encodes a putative PLC with a calculated molecular mass of 82 032 Da and an isoelectric point of 5.93. The deduced amino acid sequence of T. cruzi PLC exhibited 23–42% overall identities with the PLCs from other organisms. Among them, PLC from Ictalurus punctatus revealed the highest identity to T. cruzi PLC. The percentage identities of the entire proteins and the catalytic X/Y domains suggested that T. cruzi PLC is more evolutionarily related to the PLCs of higher eukaryotes than to those of lower unicellular eukaryotes. The tetrad analysis of the segregants of the Saccharomyces cerevisiae PLC1/plc1::HIS3 diploid strain transformed with the T. cruzi PLC-expressing plasmid showed that expression of T. cruzi PLC suppressed the growth defect caused by the plc1 disruption in yeasts. Temperature-sensitive phenotype of the S. cerevisiae plc1-mutant haploid strain was also suppressed by the expression of T. cruzi PLC. The phosphatidyl inositol-4,5-biphosphate (PtdIns(4,5)P 2) hydrolyzing activity of T. cruzi PLC was demonstrated in the lysate from the plc1-temperature sensitive yeast mutant strain transformed with the T. cruzi PLC-expressing plasmid. The yeast-expressed T. cruzi PLC showed an absolute Ca 2+ dependence which was similar to mammalian PLC isoforms: the half-maximal activity at 0.5–1×10 −5 M Ca 2+ and the maximal activity at 1–2×10 −4 M Ca 2+.

Immunocytochemical localization of phospholipase C isozymes in cord blood and adult T-lymphocytes.

The response of T-cells to peptide antigen plus major histocompatibility complex (MHC) consists of a series of cellular events collectively called T-cell activation. An essential component of this pathway is phospholipase C (PLC)gamma1, whose hydrolytic activity increases rapidly after binding of ligands to the T-cell receptor (TCR) and consequent activation of tyrosine kinases. Recent studies also suggest a GTP binding protein-dependent activation of PLCbeta during the early steps of T-cell activation. On the basis of these findings, we first checked the expression of PLC isoforms by Western blotting and by confocal and electron microscopy techniques, and then we looked for the phosphoinositide breakdown induced by CD3 engagement in cord and adult T-lymphocytes. Our results indicated that PLCbeta1 was almost exclusively expressed in cord T-cells, whereas PLCbeta2 was more strongly represented in the adult. The amount of PLCgamma1 was found to be larger in the adult than in cord cells. No significant differences were found in PLCgamma2 and delta2 expression. PLCdelta1 was scarcely detectable. On CD3 stimulation, adult lymphocytes gave rise, as expected, to a dramatic increase in phosphoinositide breakdown, whereas in cord cells this response was scarcely detected. These results indicate that a shift in PLC expression occurs in the postnatal period and that this change is associated with induction of the capability to respond to CD3 engagement with phosphoinositide hydrolysis.

G-protein signalling pathways and oestrogen: a role of balanced maintenance in osteoblasts

Oestrogen (E 2) is an important regulator of bone cell function and alterations in oestrogen levels may cause abnormal bone metabolism in vivo. In this study we examined the long term effects of 17β-oestradiol (17β-E 2) on G-proteins and the secondary signalling pathways of phospholipase C (PLC), cyclic adenosine monophosphate (cAMP), and 1,4,5-inositol triphosphate (IP 3). Cells from neonatal mouse calvariae were cultured in phenol red-free RPMI 1640 medium supplemented with charcoal stripped foetal calf serum for 192 h with either oestrogen (10 −8 M), or oestrogen withdrawal after 48 h. Cultures were stimulated for the final 48 h with IL-6 (10 −10 M), or left unstimulated. Western blot analysis was undertaken on osteoblast membrane preparations obtained by 10 mM Tris-HCl, 0.1 mM EDTA pH 7.8 and centrifugation at 40 000× g for 2 h. For cAMP study, cells were stimulated with IL-6 for either 15 min or 30 min. Intracellular cAMP was extracted from cells and measured by ELISA methodology. For the IP 3 assay, cells were stimulated with IL-6 for 20 s and IP 3 levels measured using radioimmunoassay. The blots revealed increased levels of G iα-, and G qα-proteins with oestrogen withdrawal and IL-6 stimulation. This was in comparison to cells which were unstimulated, or stimulated with IL-6 with continuous 17β-E 2, or IL-6 alone. G sα expression decreased with oestrogen withdrawal compared to the control. Limited amounts of G iα-, G sα-, and G qα-proteins were identified with continuous 17β-E 2. The levels of PLC isoforms PLCβ 1-2 were not affected by the differing oestrogen conditions. The cAMP production induced by IL-6 stimulation for 30 min and withdrawal of 17β-E 2 was lower and significantly different compared to the control study ( P<0.05). Also IL-6 activation with continuous oestradiol increased cAMP levels and was significantly different from the control cells ( P<0.01). However, 17β-E 2 had no effect on the formation of intracellular IP 3, although IL-6 significantly lowered IP 3 levels in all the groups compared to the control ( P<0.01). These results suggest that oestrogen modulates the signal transduction pathways of G-protein molecules, and the secondary pathways of cAMP in mouse osteoblast-like cells.

Immunolocalization of phospholipase C isoforms in rat kidney

Phospholipase C (PLC) is an important factor in signal transduction because this enzyme is activated by several hormones and growth factors. Eight PLC isoforms have been described raising the possibility that different cells express a single isoform or activate specific isoforms in different cells. Therefore, the goal of this study was to determine which PLC isoforms are expressed in specific regions of rat kidney. Western blot analysis was performed in microdissected nephron segments of rat kidney, while immunohistochemical analysis was performed on whole rat kidney slices using PLC isoform-specific antibodies. All three families of PLC isoforms (beta, gamma, and delta) were present throughout the cortical and medullary regions of the kidney. Only the PLC-beta1 isoform was observed in the brush border of the proximal tubule, but all isoforms were present in glomeruli and in the cytoplasm of tubular epithelial cells. In addition, only the PLC-gamma1 isoform was expressed in the internal elastic lamina of the renal artery, while vasa recta expressed PLC-beta1 most intensely. Medullary thick ascending limbs showed an intense level of expression of all three isoforms. Multiple PLC isoforms are present in glomeruli, renal tubules, and renal vasculature in vivo, but with some segment-specific differences. These findings suggest that the response of a specific cell is not determined by expression of only one PLC isoform, with the exception of the brush border of the proximal tubule and the renal arteries. Instead, the presence of multiple PLC isoforms in specific regions of the kidney suggests that hormonal regulation in vivo involves mechanisms beyond cell-specific isoforms of PLC.

Involvement of phospholipase Cgamma1 in mouse egg activation induced by a truncated form of the C-kit tyrosine kinase present in spermatozoa.

Microinjection of a truncated form of the c-kit tyrosine kinase present in mouse spermatozoa (tr-kit) activates mouse eggs parthenogenetically, and tr-kit- induced egg activation is inhibited by preincubation with an inhibitor of phospholipase C (PLC) (Sette, C., A. Bevilacqua, A. Bianchini, F. Mangia, R. Geremia, and P. Rossi. 1997. Development [Camb.]. 124:2267-2274). Co-injection of glutathione-S-transferase (GST) fusion proteins containing the src-homology (SH) domains of the gamma1 isoform of PLC (PLCgamma1) competitively inhibits tr-kit- induced egg activation. A GST fusion protein containing the SH3 domain of PLCgamma1 inhibits egg activation as efficiently as the whole SH region, while a GST fusion protein containing the two SH2 domains is much less effective. A GST fusion protein containing the SH3 domain of the Grb2 adaptor protein does not inhibit tr-kit-induced egg activation, showing that the effect of the SH3 domain of PLCgamma1 is specific. Tr-kit-induced egg activation is also suppressed by co-injection of antibodies raised against the PLCgamma1 SH domains, but not against the PLCgamma1 COOH-terminal region. In transfected COS cells, coexpression of PLCgamma1 and tr-kit increases diacylglycerol and inositol phosphate production, and the phosphotyrosine content of PLCgamma1 with respect to cells expressing PLCgamma1 alone. These data indicate that tr-kit activates PLCgamma1, and that the SH3 domain of PLCgamma1 is essential for tr-kit-induced egg activation.

Pancreastatin activates β3 isoform of phospholipase C via G α11 protein stimulation in rat liver membranes

Pancreastatin (PST) receptors have been recently shown to mediate activation of phospholipase C (PLC) in rat liver membranes. There is evidence that the G protein that links pancreastatin receptor with PLC- β is pertussis toxin-insensitive and belongs to the G α q family. Here, we have employed blocking antisera to sort out the specific PLC- β isoform as well as the specific G α subunit activated by PST receptor in rat liver membranes. The presence of different PLC- β isoforms was checked by immunoblot analysis. Only PLC- β4 was not detected, whereas PLC- β1, β2 and β3 were abundant in rat liver membranes. However, only anti-PLC- β3 serum was able to block the PST receptor response. We also checked the expression of G α q and G α 11 in rat liver membranes by immunoblot. Even though both isoforms were present, only anti-G α 11 serum was able to block the PST receptor response. In order to check the specificity of the blocking antisera, we employed them to block the effect of ADP and thrombin stimulating PLC activity in platelet membranes, a system lacking G α 11. Anti-G α q but not anti-G α 11 sera were able to block the agonist stimulated PLC activity. These data suggest that PST receptor response is mediated by the activation of the β3 isoform of PLC via G α 11 protein stimulation in rat liver membranes.

Phospholipase C β and Membrane Action of Calcitriol and Estradiol

We have shown that estrogens and calcitriol, the hormonally active form of vitamin D, increase the concentration of intracellular calcium ([Ca2+]i) within 5 s by mobilizing calcium from the endoplasmic reticulum and the formation of inositol 1,4, 5-trisphosphate and diacylglycerol. Because the activation of effectors as phospholipase C (PLC) coupled to G-proteins is the early event in the signal transduction pathway leading to the inositol 1,4,5-trisphosphate formation and to [Ca2+]i increase, we described different PLC isoforms (beta1, beta2, gamma1, and gamma2, but not beta4) in female rat osteoblasts using Western immunoblotting. The data showed that phospholipase C beta was involved in the mobilization of Ca2+ from the endoplasmic reticulum of Fura-2-loaded confluent osteoblasts by calcitriol and 17beta estradiol, and PLC gamma was ineffective. The data also showed that only a PLC beta1 linked to a Pertussis toxin-insensitive G-protein and a PLC beta2 coupled to a Pertussis toxin-sensitive G-protein are involved in the effects of calcitriol and 17beta estradiol on the mobilization of Ca2+ from intracellular Ca2+ stores. In conclusion, these results may be an important step toward understanding membrane effects of these steroids and may be an additional argument in favor of membrane receptors to steroid hormones.