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Z Naor

Publications and source records attributed to Z Naor.

At least 73 records · Page 4Linked to original sources
PubMed1990

Further studies on the involvement of protein kinase C in human sperm flagellar motility.

Addition of the phorbol ester 12-O-tetradecanoyl phorbol-13-acetate (TPA) or the membrane-permeable diacylglycerol analog, 1-oleoyl-2-acetylglycerol to human sperm resulted in increased motility. The biologically inactive 4 alpha-phorbol 12,13 didecanoate had no effect on flagellar motility. Basal motility was markedly reduced in the absence of Ca2+ in the incubation medium, but TPA-induced sperm motility persisted even in the absence of Ca2+. Sperm motility was also enhanced by the Ca2+ ionophore ionomycin in a Ca2(+)-dependent, protein kinase c (PKC)-independent fashion. Although all stimulants examined here reached maximal response at about 15 min of incubation, nevertheless whereas the effect of TPA and 1-oleoyl-2-acetylglycerol declined at 60 min of incubation, that of ionomycin still persisted. Human sperm PKC activity is extremely low and represents only about 20% and 25% of the specific activity recovered from PC-12 and rat pituitary cells, respectively. Immunohistochemical analysis using various type-specific PKC antibodies revealed staining only in the equatorial segment and the principal piece of the tail. Thus, PKC is present in human ejaculated sperm and is involved in flagellar motility.

Adult↗
PubMed1989

Effect of guanine nucleotides on phospholipase C activity in permeabilized pituitary cells: possible involvement of an inhibitory GTP-binding protein.

Cultured pituitary cells prelabeled with myo-[2-3H] inositol were permeabilized by ATP4-, exposed to guanine nucleotides and resealed by Mg2+. Addition of guanosine 5'-0-(3-thio triphosphate) (GTP gamma S) to permeabilized cells, or gonadotropin releasing hormone (GnRH) to resealed cells, resulted in enhanced phospholipase C activity as determined by [3H] inositol phosphate (Ins-P) production. The effect was not additive, but the combined effect was partially inhibited by guanosine 5'-0-(2-thiodiphosphate) (GDP beta S) or by neomycin. Surprisingly, addition of GDP beta S (100-600 microM) on its own resulted in a dose-related increase in [3H]Ins-P accumulation. Several nucleoside triphosphates stimulated phospholipase C activity in permeabilized pituitary cells with the following order: UTP greater than GTP gamma S greater than ATP greater than CTP. The stimulatory effect of UTP, ATP and CTP, but not GTP gamma S or GDP beta S, could also be demonstrated in normal pituitary cells suggesting a receptor-activated mechanism. GTP and GTP gamma S decreased the affinity of GnRH binding to pituitary membranes and stimulated LH secretion in permeabilized cells. These results suggest the existence of at least two G-proteins (stimulatory and inhibitory) which are involved in phospholipase C activation and GnRH action in pituitary cells.

Animals↗
PubMed1989

Selective activation of the gamma-subspecies of protein kinase C from bovine cerebellum by arachidonic acid and its lipoxygenase metabolites.

The gamma-subspecies of protein kinase C (PKC) apparently is expressed only in central nervous tissues, and at a high level in the cerebellum and hippocampus. gamma-PKC from bovine cerebellum, but not the alpha- or beta I/beta II-subspecies, is activated by micromolar concentrations of arachidonic acid (AA), in the absence of both phospholipid and diacylglycerol. A significant component of this activation is also calcium independent. Other unsaturated fatty acids are much less active in this respect. Among the AA metabolites tested, lipoxin A (5(S),6(R),15(S)-11-cis-isomer) was a potent, selective activator of the gamma-subspecies, and also, to a lesser extent, 12(S)-hydroxy-5,8,10,14-eicosatetraenoic acid could support activation. These results raise the possibility that AA and some of its lipoxygenase metabolites may function as messenger molecules in neurones to activate the gamma-subspecies of PKC.

Animals↗
PubMed1989

Induction of exocytosis in permeabilized pituitary cells by alpha- and beta-type protein kinase C.

Protein kinase C is now recognized to comprise a family of closely related subspecies (PKCs). When cultured rat pituitary cells were permeabilized by digitonin for 5 min in the absence of Ca2+, endogenous PKC activity was decreased by 72%. PKC depletion was also achieved by prior treatment (24 hr) with high concentrations of phorbol 12-myristate 13-acetate (PMA). When purified activated brain PKCs were added for 30 min to PMA-pretreated, digitonin-permeabilized cells, only alpha- and beta- but not gamma-type PKC stimulated luteinizing hormone release. Since PKC was implicated as a mediator of gonadotropin secretion, gonadotropin-releasing hormone might utilize alpha- and beta-type PKCs for stimulation of gonadotropin secretion; alpha- and beta-type PKCs might participate also in other exocytotic responses in diverse biological systems in which PKC was implicated.

Animals↗
PubMed1988

The heterogeneity and differential expression of protein kinase C in nervous tissues.

Protein kinase C exists as a large family of multiple subspecies with subtle individual characteristics. This heterogeneity comes from different genes as well as from different splicings of a single RNA transcript. The members of this family have closely related structures with a high degree of homology. Biochemical studies have shown that their mode of activation and kinetic and catalytic properties differ slightly from one another. By using a combination of biochemical and immunocytochemical techniques, their differential regional and cellular expression have been shown in the nervous tissues. Each member of this enzyme family may have a specialized function in transducing various physiological and pathological signals into different cell types.

Amino Acid Sequence↗
PubMed1988

Calcium-independent activation of hypothalamic type I protein kinase C by unsaturated fatty acids.

Among multiple subspecies of the protein kinase C (PKC) family, type I PKC from the hypothalamus, having the structure related to the gamma-sequence, responds to low concentrations of arachidonic acid to exhibit marked enzymatic activity. This mode of activation does not require elevated Ca2+ levels, nor does it depend on diacylglycerol and phospholipid. Type I PKC is expressed only in limited regions of central nervous tissues, such as the hypothalamus. This PKC subspecies is not detected in the pituitary gland. The results suggest that the activation of type I PKC may not always be directly associated with inositol phospholipid hydrolysis, and that this subspecies may play a role in the modulation of specialized functions of the hypothalamus.

Animals↗
PubMed1988

Gonadotropin-releasing hormone-induced rise in cytosolic free Ca2+ levels: mobilization of cellular and extracellular Ca2+ pools and relationship to gonadotropin secretion.

Addition of GnRH to pituitary gonadotrophs preloaded with Quin 2 resulted in a rapid (approximately 8 s) mobilization of an ionomycin-sensitive intracellular Ca2+ pool. A second component of Ca2+ entry via voltage dependent channels contributed about 45% of the peak cytosolic free Ca2+ concentration ([Ca2+]i). Thereafter, influx of Ca2+ via voltage-sensitive and -insensitive channels is responsible for maintenance of elevated [Ca2+]i during the second phase of GnRH action. Addition of inositol 1,4,5-trisphosphate (IP3) to permeabilized pituitary cells resulted in a Ca2+ transient, released from a nonmitochondrial pool, which maintained ambient free Ca2+ concentration around 170 nM in an ATP-dependent mechanism. Successive stimulations of the cells with IP3 produced an attenuated response. Elevation of the gonadotroph [Ca2+]i by ionomycin, to levels equivalent to that induced by GnRH, resulted in LH release amounting to only 45% of the response to the neurohormone. Activation of the voltage-dependent Ca2+ channels by the dihydropyridine Ca2+-agonist [methyl 1,4-dihydro-2,6-dimethyl-3-nitro-4-(2-trifluoromethylphenyl)-pyridine- 5-carboxylate (BAYK8644)] stimulated LH release, 36% of the GnRH (100 nM) response being reached by 10(-8) M of the drug, both [Ca2+]i elevation and GnRH-induced LH release were inhibited similarly (40-50%) by the dihydropyridine Ca2+-antagonist nifedipine. The results indicate that peak [Ca2+]i induced by GnRH in pituitary gonadotrophs is derived mainly from ionomycin-sensitive cellular stores most likely via IP3 formation.(ABSTRACT TRUNCATED AT 250 WORDS)

3-Pyridinecarboxylic acid, 1,4-dihydro-2,6-dimethy↗
PubMed1987

The initial phase of GnRH-stimulated LH release from pituitary cells is independent of calcium entry through voltage-gated channels.

Kinetic studies on gonadotropin-releasing hormone (GnRH)-stimulated luteinizing hormone (LH) release were undertaken using rat and chicken pituitary cell cultures. In response to continuous GnRH stimulation, a biphasic pattern of LH release was demonstrated. The two phases showed different susceptibility to the voltage-gated Ca2+ channel blockers D600 and nifedipine. The first (transient) phase of LH release was unaffected by the Ca2+ channel blockers whereas the second (sustained) phase was inhibited by both drugs. These results indicate that the initial phase of LH release is independent of Ca2+ entry through voltage-gated Ca2+ channels and may depend on mobilisation of intracellular Ca2+ or entry of extracellular Ca2+ through another mechanism.

Animals↗
PubMed1987

Effect of phorbol ester on stimulus-secretion coupling mechanisms in gonadotropin releasing hormone-stimulated pituitary gonadotrophs.

The feedback regulatory control mechanism exerted by activated Ca2+/phospholipid-dependent protein C kinase upon gonadotropin releasing hormone (GnRH) binding, stimulation of phosphoinositide turnover and gonadotropin secretion was investigated in cultured pituitary cells. Addition of the tumor promoter phorbol ester 12-O-tetradecanoylphorbol-13-acetate (TPA), at concentrations which activate pituitary protein C kinase, to cultured pituitary cells resulted in up-regulation of GnRH receptors (155% at 4 h). The stimulatory effect of GnRH on [3H]inositol phosphates (Ins-P) production in myo-[2-3H]inositol prelabeled pituitary cells was not inhibited by prior treatment of the cells with TPA (10(-9)-10(-7) M). Higher concentrations of TPA (10(-6)-10(-5) M) inhibited the effect of GnRH on [3H]Ins-P production. Increasing concentrations of TPA or the permeable analog of diacylglycerol 1-oleoyl-2-acetylglycerol (OAG) stimulated luteinizing hormone (LH) release from cultured pituitary cells with ED50 values of 5 x 10(-9) M and 10 micrograms/ml, respectively. No consistent inhibition or additivity of LH release was observed when increasing doses of TPA or OAG were added with a submaximal dose of GnRH. These results suggest that protein C kinase might mediate the known homologous up-regulation of GnRH receptors during the reproductive cycle. Protein C kinase is positively involved in mediating the process of gonadotropin secretion. Unlike many other systems, activation of protein C kinase in pituitary gonadotrophs is not involved in negative feed-back regulation of stimulus-secretion-coupling mechanisms in GnRH-stimulated gonadotrophs.

Animals↗
PubMed1987

Differential expression of multiple protein kinase C subspecies in rat central nervous tissue.

Protein kinase C from a number of areas of rat central nervous tissue was resolved into three distinct fractions upon hydroxyapatite column chromatography. One of the enzyme fractions, designated type II, could be further distinguished into two subspecies with polyclonal antisera, which were raised against synthetic peptides specific for the predicted amino acid sequences of two alternative cDNA clones encoding this enzyme type. Using a combination of these biochemical and immunological techniques, the relative activity of the multiple subspecies of protein kinase C was assessed for each brain area. A distinct regional pattern of expression was found, which per se may be an important factor in determining the response of different neuronal cell types to extracellular stimuli.

Animals↗
PubMed1987

Distribution and activation of protein kinase C in the rat testis tissue.

The distribution and role of the calcium-activated, phospholipid-dependent protein kinase C (PK-C) was studied in rat testis. When testis tissue was homogenized in the presence of 2 mmol/l EDTA and EGTA, the majority (greater than 70%) of the PK-C activity was soluble, the rest was released from the particulate fraction by solubilization with 0.3% Triton X-100. Without chelating agents the soluble PK-C activity was undetectable, and only partially recovered from solubilized membranes. Preincubation of the tissue with the tumor-promoting phorbol ester 12-O-tetradecanoylphorbol-13-acetate (TPA, 10(-7) mol/l) translocated PK-C to the membranes, and the majority of this activity was recovered by solubilization. Mobility of testicular soluble PK-C activity in HPLC-DEAE cellulose chromatography was similar to that of the brain enzyme. This single step purified testicular PK-C activity 140-fold. The specific activity and subcellular distribution of PK-C was similar in whole testis tissue and separated seminiferous tubules (160-210 pmol 32P X mg protein-1 X min-1 in the soluble and particulate fractions), but 2- to 3-fold higher in purified Leydig cells. However, the majority of total testicular PK-C activity appeared to be of tubular origin. Unilateral cryptorchidism for 1 week reduced PK-C of the abdominal testis by 50%, and the activity of dissected seminiferous tubules varied according to the epithelial wave. Both findings suggest that the bulk of the activity resides in the seminiferous epithelium. Involvement of PK-C in Leydig cell function was demonstrated using the TPA, which at 10(-7) mol/l inhibited basal cAMP production by 50% (P less than 0.01) but increased that of testosterone by 2- to 3-fold (P less than 0.01). On the other hand, when incubated with hCG, TPA inhibited both cAMP and testosterone production; the ED50s of hCG stimulation increased 4- to 10-fold with both parameters. It is concluded that PK-C activity is present in both the seminiferous tubules and Leydig cells, and is involved in the regulation of these testicular compartments. Its total activity and subcellular distribution are at variance according to the functional state and endocrine milieu of the testis.

Animals↗
PubMed1987

Cytosolic free calcium levels in cultured pituitary cells separated by centrifugal elutriation: effect of gonadotropin-releasing hormone.

The cytosolic concentration of free Ca2+ ([Ca2+]i) in normal rat pituitary cells separated by centrifugal elutriation was monitored with the fluorescent Ca2+ indicator Quin 2. GnRH (10(-7) M) induced a rapid rise (6-8 sec) in the gonadotroph's [Ca2+]i, followed by a plateau phase of prolonged elevated [Ca2+]i which lasted about 15 min. The stimulatory effect of GnRH was dose dependent, with an ED50 of 10(-9) M, and was blocked by the potent antagonist [Dp-Glu1,pclPhe2,DTrp3.6]GnRH. GnRH elevated [Ca2+]i only in gonadotroph-enriched cell fractions, whereas TRH and GH-releasing factor (GRF) elevated [Ca2+]i in mammotroph- and somatotroph-enriched cells fractions, respectively. A rapid increase (first phase) in [Ca2+]i induced by GnRH was observed in Ca2+-free medium containing EGTA, but this rapid phase was terminated within 2 min. Readdition of Ca2+ to the medium induced a second slower rise in [Ca2+]i (plateau phase). Addition of K+ caused a rapid rise in [Ca2+]i, which was dependent on extracellular Ca2+, but was not affected by prior stimulation with GnRH. On the other hand, stimulation of gonadotroph's [Ca2+]i response by GnRH desensitized the cells to a subsequent GnRH challenge within the time frame studied. These findings indicate an elevation of [Ca2+]i induced by GnRH, TRH, and GRF in their respective separated target cells in the rat pituitary. The rise in [Ca2+]i in GnRH-stimulated gonadotrophs originates partly from intracellular Ca2+ pools and partly from influx of Ca2+ across the cell membrane.

Aminoquinolines↗
PubMed1986

Gonadotropin-releasing hormone activates a rapid Ca2+-independent phosphodiester hydrolysis of polyphosphoinositides in pituitary gonadotrophs.

Addition of gonadotropin releasing hormone (GnRH) to pituitary cells prelabeled with [32P]Pi or with myo-[2-3H]inositol, resulted in a rapid decrease in the level of [32P]phosphatidylinositol 4,5-bisphosphate (approximately 10 s), and in [32P]phosphatidylinositol 4-phosphate (approximately 1 min), followed by increased labeling of [32P]phosphatidylinositol and [32P]phosphatidic acid (1 min). GnRH stimulated the appearance of [3H]myo-inositol 1,4,5-trisphosphate (10 s), [3H]myo-inositol 1,4-bisphosphate (15 s), and [3H]myo-inositol 1-phosphate (1 min) in the presence of Li+ (10 mM). Li+ alone stimulated the accumulation of [3H]myo-inositol 1-phosphate and [3H]myo-inositol 1,4-bisphosphate but not [3H]myo-inositol 1,4,5-trisphosphate, but had no effect on luteinizing hormone release. The effect of GnRH on inositol phosphates (Ins-P) production was dose-related (ED50 = 1-5 nM), and was blocked by a potent antagonist [D-pGlu,pClPhe,D-Trp]GnRH. Elevation of cytosolic free Ca2+ levels ([Ca2+]i), by ionomycin and A23187 from intracellular or extracellular Ca2+ pools, respectively, had no significant effect on [3H]Ins-P production. GnRH-induced [3H]Ins-P production was not dependent on extracellular Ca2+ and was noticed also after extracellular or intracellular Ca2+ mobilization by A23187 or ionomycin, respectively. The effect of GnRH on [3H]Ins-P accumulation was not affected by prior treatment of the cells with the tumor promoter phorbol ester 12-O-tetradecanoylphorbol-13-acetate or with islet-activating protein pertussis toxin. These results indicate that GnRH stimulates a rapid phosphodiester hydrolysis of polyphosphoinositides. The stimulatory effect is not mediated via an islet-activating protein-substrate, is not dependent on elevation of [Ca2+]i, neither is it negatively regulated by 12-O-tetradecanoylphorbol-13-acetate which activates Ca2+/phospholipid-dependent protein C kinase. The results are consistent with the hypothesis that GnRH-induced phosphoinositide turnover is responsible for Ca2+ mobilization followed by gonadotropin release.

Animals↗
PubMed1986

Lipoxygenase inhibitor and colchicine as anti-arthritic agents in the rat.

The non-steroidal anti-inflammatory agents do not have identical activities on the various pathways of arachidonic acid metabolism. The purpose of this study is to examine and compare the activities of colchicine, an anti-arthritic agent, indomethacin, a known prostaglandin synthesis inhibitor and nordihydroguaiaretic acid (NDGA), a lipoxygenase inhibitor, in an experimental model of arthritis. Acute arthritis of the knee was induced in rats by injection of lipopolysaccharide (LPS) into the joints. Arthritis was characterized by an increase in joint diameter (18%), increased synovial weight (34%) and an increase in synovial prostaglandin E (PGE) production (56%). While administration of all of the agents examined abolished LPS-induced joint diameter and synovial weight increase, only indomethacin reduced increased PGE content. NDGA and colchicine had no inhibitory effect on LPS-induced PGE production, and moreover they actually stimulated PGE production when compared to control values. It is concluded that: Among the mediators of the inflammatory process are factors sensitive to colchicine and NDGA which are not PGs. Lipoxygenase products of arachidonic acid including leukotrienes may have an important role in inflammation. Leukotrienes and prostaglandins may act in concert in mediating the inflammatory process.

Animals↗
PubMed1986

Calcium-dependent actions of gonadotropin-releasing hormone agonist and luteinizing hormone upon cyclic AMP and progesterone production in rat ovarian granulosa cells.

The Ca2+ dependency of the direct stimulatory effect of the gonadotropin-releasing hormone (GnRH) agonist analog [D-Ser(t-Bu)6]des-Gly10-GnRH-N-ethylamide (GnRHa) on progesterone production was investigated and compared to that of luteinizing hormone (LH) in rat granulosa cells from preovulatory follicles. Removal of extracellular Ca2+ by EGTA, or the use of the Ca2+ channel blockers verapamil and La3+, resulted in complete inhibition of GnRHa-induced progesterone production and a partial inhibition of LH-stimulated progesterone production (80, 80 and 50% inhibition respectively for EGTA, verapamil and La3+). Removal of extracellular Ca2+ increased the ED50 for LH-induced cAMP production by four-fold (from 80 to 330 ng/ml) and decreased maximal nucleotide formation by 44%. LH-induced cAMP production was also inhibited partially by verapamil (35%) at 10(-4) M drug concentration. GnRHa had no effect on cAMP production in the presence or absence of Ca2+. GnRHa and LH were found to have maximal effects on progesterone production at about 0.5 mM of Ca2+ in the incubation medium. On the other hand the stimulatory effect of dibutyryl cAMP [Bu)2cAMP) on progesterone production showed little dependency on extracellular Ca2+. The calmodulin antagonist trifluoperazine (TFP) caused concentration-dependent inhibition of the stimulatory action of GnRHa and LH on progesterone production with IC50 values of 3 and 8 microM, respectively. The stimulatory effect of (Bu)2cAMP on progesterone synthesis was attenuated by verapamil and TFP. These results indicate that the direct stimulatory effect of GnRH on ovarian progesterone production is absolutely dependent on Ca2+.(ABSTRACT TRUNCATED AT 250 WORDS)

Animals↗