Identification of glycation at the N-terminus of albumin by gas chromatography-mass spectrometry.

It has previously been shown that neurotensin binds to high-affinity receptors in the adenocarcinoma HT29 cell line, and that receptor occupancy leads to inositol phosphate formation. The present study was designed to investigate further the effects of neurotensin on calcium mobilization and protein kinase C (PKC) activation in HT29 cells, and to assess the role of GTP-binding proteins (G-proteins) in the neurotensin response. Direct measurements of cytosolic Ca2+ variations using the fluorescent indicator quin 2 showed that neurotensin (0.1-1 microM) elicited Ca2+ transients in HT29 cells. These transients occurred after the neurotensin-stimulated formation of Ins(1,4,5)P3, as measured by means of a specific radioreceptor assay. In addition, the peptide induced a decrease in the 45Ca2+ content of cells previously equilibrated with this isotope. The peptide effect was rapid, long-lasting and concentration-dependent, with an EC50 of 2 nM. Phorbol 12-myristate 13-acetate (PMA) inhibited by 50% the neurotensin effects on both intracellular Ca2+ and inositol phosphate levels. The inhibition by PMA was abolished in PKC-depleted cells. Pertussis toxin had no effect on either the Ca2+ or inositol phosphate responses to neurotensin. Epidermal growth factor (EGF) receptors which are present in HT29 cells have been shown to be down-regulated through phosphorylation by PKC in a variety of systems. Here, PMA markedly (70-80%) inhibited EGF binding to HT29 cells. Scatchard analysis revealed that PMA abolished the high-affinity component of EGF binding, an effect that was totally reversed in PKC-depleted cells. In contrast, neurotensin slightly (10-20%) inhibited EGF binding to HT29 cells, and its effect was only partly reversed by PKC depletion. Neurotensin had no detectable effect on sn-1,2-diacylglycerol levels in HT29 cells, as measured by a specific and sensitive enzymic assay. In membranes prepared from HT29 cells, monoiodo[125I-Tyr3]neurotensin bound to a single population of receptors with a dissociation constant of 0.27 nM. Sodium and GTP inhibited neurotensin binding in a concentration-dependent manner. Maximal inhibition reached 80% with Na+ and 35% with GTP.IC50 values were 20 mM and 0.2 microM for Na+ and GTP respectively. Li+ and K+ were less effective than Na+ and the effects of GTP were shared by GDP and guanosine-5'-[beta gamma- imido]triphosphate but not by ATP. Scatchard analysis of binding data indicated that Na+ and GTP converted the high-affinity neurotensin-binding sites into lower affinity binding sites. The properties of the effects of Na+ and GTP on neurotensin-receptor interactions are characteristic of those receptors which interact with G-proteins.(ABSTRACT TRUNCATED AT 400 WORDS)     

Effect of ethanol on receptor-stimulated phosphatidic acid and polyphosphoinositide metabolism in mouse brain

The effects of chronic ethanol consumption as well as the effects of ethanol added in vitro on phosphoinositide metabolism were determined in mouse forebrain. [32P] incorporation into synaptosomal phosphatidic acid (PhA) was stimulated through both M1 muscarinic cholinergic and alpha 1 adrenergic receptor activation. Similarly, [3H]inositol 1-PO4 accumulation in brain slices was stimulated through these same receptors, but could also be stimulated by histamine1 receptor activation. In mice made physically dependent to ethanol, the magnitude of receptor-mediated [32P] incorporation in PhA did not differ from that of control animals. However, ethanol (100mM) added in vitro to synaptosomes from control mice significantly inhibited the carbamylcholine stimulated PhA response, but had no effect on the response to norepinephrine. Carbamylcholine stimulated [32P] incorporation into PhA, however, was no longer significantly inhibited by the addition of 100mM ethanol to synaptosomes from physically dependent-tolerant animals indicating that a cellular tolerance had developed. In contrast, receptor mediated [3H]inositol 1-PO4 accumulation in brain slices was not significantly affected by either chronic ethanol treatment or the in vitro addition of ethanol as high as 200mM. It is concluded that the muscarinic cholinergic stimulation of [32P] incorporation into PhA, but not [3H]inositol 1-PO4 accumulation is relatively more sensitive to the direct effects of ethanol than are the other receptor mediated phospholipid responses examined in the present investigation and that this sensitivity is lost in animals made behaviorally tolerant and physically dependent to ethanol.     

Short chain alcohols activate guanine nucleotide-dependent phosphoinositidase C in turkey erythrocyte membranes

The ability of alcohols to regulate inositol lipid-specific phospholipase C (phosphoinositidase C) was examined in turkey erythrocyte ghosts prepared by cell lysis of erythrocytes which were prelabeled with [3H] inositol. Guanosine 5'-[gamma-thiotriphosphate] GTP[S] stimulated the production of both [3H]inositol bisphosphate (18-fold) and [3H]inositol trisphosphate (6-fold) in this system. The accumulation of [3H]inositol bisphosphate and [3H]inositol trisphosphate was linear up to 8 min following an initial lag period of 1-2 min. Ethanol (300 mM) reduced the lag period for [3H]inositol phosphate accumulation at submaximal GTP[S] concentrations and caused a shift to the left (3-fold) in the dose-response curve. Other short chain alcohols, methanol (300 mM), 1-propanol (200 mM), and 1-butanol (50 mM) also enhanced the accumulation of [3H] inositol phosphates in the presence of submaximal GTP[S] concentrations. Receptor activation by the purinergic agonist adenosine 5'-[beta-thio]disphosphate (ADP[S]) (10 microM) also reduced the lag period for [3H] inositol phosphate formation and shifted the GTP[S] dose response to the left (10-fold). In addition, ADP[S] increased the response to maximal GTP[S] concentrations. The formation of [3H]inositol phosphates induced by GTP[S] was associated with a concomitant decrease in labeling of both [3H]phosphatidylinositol monophosphate and [3H]phosphatidylinositol bisphosphate, but no decrease in [3H]phosphatidylinositol was observed. All of the alcohols tested enhanced the breakdown of [3H]polyphosphoinositides in the presence of GTP[S]. The dose response to guanosine 5'-[beta gamma-imino]triphosphate for [3H]inositol phosphate formation was displaced to the left by ethanol (300 mM) and ADP[S] (10 microM) (2- and 7-fold), respectively. ADP[S] also enhanced the maximal response to guanosine 5'-[beta gamma-imino]triphosphate. The [3H]inositol phosphate formation produced in response to NaF was unaffected by either ethanol or receptor activation. These results indicate that alcohols initiate an activation of phosphoinositidase C, mediated at the level of the regulatory guanine nucleotide-binding protein.     

G Proteins Coupled to Phospholipase C: Molecular Targets of Long-Term Ethanol Exposure

Long-term ethanol exposure is known to inhibit bradykinin-stimulated phosphoinositide hydrolysis in cultures of neuroblastoma x glioma 108-15 cells. In the present study, [3H]bradykinin binding, GTP-binding protein function, and phospholipase C activity were assayed in cells grown for 4 days in 100 mM ethanol with the aim of elucidating the molecular target of ethanol on signal transduction coupled to inositol trisphosphate and diacylglycerol formation. Ethanol exposure reduced guanosine 5'-O-(3-thiotriphosphate) [GTP(S)]- and, to a lesser extent, NaF/AlCl3-stimulated phosphoinositide hydrolysis, whereas it had no effect on the enzymatic activity of a phosphatidylinositol 4,5-bisphosphate-specific phospholipase C. [3H]Bradykinin binding in the absence of GTP(S) was not influenced by ethanol exposure. However, the reduction in [3H]bradykinin binding seen in control cells after addition of GTP analogue was inhibited in cells grown in ethanol-containing medium. The results indicate that long-term ethanol exposure exerts its effects on receptor-stimulated phosphoinositide hydrolysis primarily at the level of the GTP-binding protein.     

Activation of B2 bradykinin receptors by neurotensin

Many previous reports suggested that relatively high concentrations of neurotensin were required to exert its effects on neurotransmitter secretion. The neurotensin binding sites, which recognize high concentrations of neurotensin, were characterized in rat pheochromocytoma (PC12) cells. When PC12 cells were treated with neurotensin, [3H]norepinephrine secretion and elevation of cytosolic calcium were evoked at EC(50) values of 59+/-4 and 37+/-7 microM, respectively. Both calcium release and inositol 1,4,5-trisphosphate (IP(3)) production induced by neurotensin suggested involvement of phospholipase C. Experiments with simultaneous or sequential treatment with neurotensin and bradykinin suggested that neurotensin and bradykinin act on the same binding sites. Furthermore, both inhibition of bradykinin- and neurotensin-induced calcium rises by bradykinin receptor antagonists with similar IC(50) values and receptor binding analysis using [3H]bradykinin confirmed that neurotensin directly binds to B2 bradykinin receptors. The data suggest that neurotensin binds and activates the B2 bradykinin receptors.     

Stimulatory and Inhibitory Effects of N-Methyl-D-Aspartate on 3H-Inositol Polyphosphate Accumulation in Rat Cortical Slices

The actions of the excitatory amino acid N-methyl-D-aspartate (NMDA) on the accumulation of 3H-inositol polyphosphate isomers in rat cerebral cortex slices have been examined over short (less than 5 min) incubation periods. NMDA caused the dose-dependent accumulation of only [3H]inositol monophosphate and [3H]inositol bisphosphate (maximal effect between 0.3 and 1 mM), with no increase in [3H]inositol trisphosphate ([3H]InsP3) and [3H]inositol tetrakisphosphate ([3H]InsP4). HPLC analysis confirmed this, showing no increases in the breakdown products of [3H]Ins(1,3,4,5)P4. When present with the muscarinic agonist carbachol (1 mM), high concentrations of NMDA (1 mM) could almost totally inhibit carbachol-induced accumulation of 3H-inositol polyphosphates. In contrast, at lower concentrations of NMDA (10 microM), the inhibitory effect was replaced with a synergistic accumulation of inositol polyphosphates, especially [3H]InsP4 and [3H]InsP3. The inhibitory effects of NMDA were only apparent when extracellular Ca2+ was present, although incubation in media with no added Ca2+ resulted in somewhat reduced stimulatory responses to NMDA alone, but suppressed totally the inhibitory effects of 1 mM NMDA and reduced the synergistic effects of 10 microM NMDA on carbachol responses. These studies, therefore, reveal Ca(2+)-dependent effects of NMDA indicative of indirect mechanisms of action and show that care must be made in interpreting the effects of NMDA on phosphoinositide metabolism unless the inositol polyphosphate composition has been fully characterised.     

Neurotensin Stimulates Inositol Phospholipid Metabolism and Calcium Mobilization in Murine Neuroblastoma Clone N1E-115

Murine neuroblastoma cells (clone N1E-115) possess neurotensin receptors that mediate cyclic GMP synthesis. Because of the hypothesized relationship between phospholipid metabolism, intracellular Ca2+, and cyclic GMP synthesis, we determined with these cells the effects of neurotensin on 32P labeling of phospholipids, release of inositol phosphates, and intracellular Ca2+ (as determined with the use of Quin-2, a fluorescent probe sensitive to free Ca2+ levels). Neurotensin stimulated incorporation of 32P into phospholipids, especially phosphatidylinositol and phosphatidate. Neurotensin also stimulated the release of [3H]inositol phosphates with an EC50 of about 1 nM. Mean basal Ca2+ concentration in these cells was 134 nM and this level was increased in a rapid and dose-dependent manner by neurotensin, with an EC50 of 4 nM. Since the EC50 for neurotensin in stimulating cyclic GMP synthesis is 1.5 nM and the KD for binding of [3H]neurotensin at 0 degrees C is 11 nM, all these different effects appear to be shared proximal consequences of neurotensin receptor activation.     

Polyamines inhibit phospholipase C-catalysed polyphosphoinositide hydrolysis. Studies with permeabilized GH3 cells.

[3H]Inositol-labelled GH3 rat anterior pituitary tumour cells were permeabilized with digitonin and were incubated at 37 degrees C in the presence of ATP and Mg2+. [3H]Polyphosphoinositide breakdown and [3H]inositol phosphate production were stimulated by hydrolysis-resistant GTP analogues and by Ca2+. Of the nucleotides tested, guanosine 5'-[gamma-thio]triphosphate (GTP gamma S) was the most effective stimulus. Activation by GTP gamma S appeared to be mediated by a guanine nucleotide-binding (G) protein as GTP gamma S-stimulated [3H]inositol phosphate production was inhibited by other nucleotides with a potency order of GTP = GDP = guanosine 5'-[beta-thio]diphosphate greater than ITP greater than GMP greater than UTP = CTP = adenosine 5'-[gamma-thio]triphosphate. The stimulatory effects of 10 microM-GTP gamma S on [3H]inositol phosphate levels were reversed by spermine and spermidine with IC50 values of approx. 0.25 and 2 mM respectively. Putrescine was inhibitory only at higher concentrations. Similarly, GTP gamma S-induced decreases in [3H]polyphosphoinositide levels were reversed by 2.5 mM-spermine. The inhibitory effects of spermine were not overcome by supramaximal concentrations of GTP gamma S. In contrast, [3H]inositol phosphate production stimulated by addition of 0.3-0.6 mM-Ca2+ to incubation media was only partially inhibited by spermine (5 mM), and spermine was not inhibitory when added Ca2+ was increased to 1 mM. These data show that polyamines, particularly spermine, inhibit phospholipase C-catalysed polyphosphoinositide hydrolysis with a marked selectivity towards the stimulatory effects of GTP gamma S.     

Alteration of neurotensin receptors in MPTP-treated mice.

We examined the sequential changes in neurotensin receptors in the striatum and substantia nigra of mouse brains lesioned with 1-methyl-4-phenyl-1,2,3,6-tetrahydropyridine (MPTP) by receptor autoradiography, in comparison with the alterations in dopamine uptake sites. The mice received four intraperitoneal injections of MPTP (10 mg/kg) at 1-h intervals and then the brains were analyzed at 6 h and 1, 3, 7, and 21 days after the treatments. [3H]Neurotensin and [3H]mazindol were used to label neurotensin receptors and dopamine uptake sites, respectively. [3H]Neurotensin binding was significantly decreased in the striatum from 6 h to 21 days after MPTP treatment. In the substantia nigra, pars reticulata also showed a significant decrease in [3H]neurotensin binding from 3 to 21 days post-MPTP treatment. However, no significant change in [3H]neurotensin binding was observed in the pars compacta even after 21 days. On the other hand, [3H]mazindol binding was markedly decreased in the striatum and substantia nigra from 6 h to 21 days after MPTP treatment. These results indicate that neurotoxin MPTP can produce a severe decrease in neurotensin receptors and dopamine uptake sites in the striatum and substantia nigra of mice. Thus, our findings provide evidence that the dysfunction in neurotensin receptors may be involved in the degenerative processes causing Parkinson's disease.     

A role for calcium in the breakdown of inositol phospholipids in intact and digitonin-permeabilized pancreatic islets.

Glucose (20 mM) and 4-methyl-2-oxopentanoate (10 mM) both caused a pronounced stimulation of insulin release and of [3H]inositol phosphate production in rat pancreatic islets prelabelled with myo-[3H]inositol. Secretory responses to these nutrients were markedly impaired by lowering the Ca2+ concentration of the incubation medium to 10(-4)M or less, whereas stimulated inositol phosphate production was sensitive to Ca2+ within the range 10(-6)-10(-4)M. Inositol phosphate formation in response to carbamoylcholine was also found to be dependent on the presence of 10(-5)M-Ca2+ or above. Raising the concentration of K+ in the medium resulted in a progressive, Ca2+-dependent stimulation of inositol phosphate production in islets, although no significant stimulation of insulin release was observed. In islets prelabelled with myo[3H]inositol, then permeabilized by exposure to digitonin, [3H]inositol phosphate production could be triggered by raising the Ca2+ concentration from 10(-7) to 10(-5)M. This effect was dependent on the concentration of ATP and the presence of Li+, and involved detectable increases in the levels of InsP3 and InsP2 as well as InsP. A potentiation of inositol phosphate production by carbamoylcholine was observed in permeabilized islets at lower Ca2+ concentrations, although nutrient stimuli were ineffective. No significant effects were observed with guanine nucleotides or with neomycin, although NADH produced a modest increase and adriamycin a small inhibition of inositol phosphate production in permeabilized islets. These results strongly suggest that Ca2+ ions play an important role in the stimulation of inositol lipid metabolism in islets in response to nutrient secretagogues, and that inositide breakdown may actually be triggered by Ca2+ entry into the islet cells.     

Specific vasopressin binding to rat adrenal glomerulosa cells. Relationship to inositol lipid breakdown.

Cells from the zona glomerulosa of rat adrenals were isolated and maintained for 3 days in primary culture. Specific vasopressin binding was determined by using [3H]vasopressin. [3H]Vasopressin binding was time-dependent (half-time of about 2 min for 6 nM free ligand) and reversible on addition of unlabelled vasopressin (80% dissociation within 30 min). Dose-dependent [3H]vasopressin binding at equilibrium indicated that vasopressin interacted with two populations of sites: high-affinity sites (dissociation constant, Kd = 1.8 nM; maximal binding capacity = 10 fmol/10(6) cells) and low-affinity sites. Vasopressin increased the cellular content of labelled inositol mono-, bis- and tris-phosphate in cells prelabelled with myo-[3H]inositol. The vasopressin concentration eliciting half-maximal inositol phosphate accumulation was very close to the Kd value for vasopressin binding to high-affinity sites. Competition experiments using agonists and antagonists with enhanced selectivity for previously characterized vasopressin receptors indicated that vasopressin receptors from rat glomerulosa cells are V1 receptors of the vascular or hepatic subtype. The detected specific vasopressin-binding sites might represent the specific receptors mediating the mitogenic and steroidogenic effects of vasopressin on glomerulosa cells from rat adrenals.     

Metabolism of inositol phosphates in parotid cells: implications for the pathway of the phosphoinositide effect and for the possible messenger role of inositol trisphosphate

Rat parotid acinar cells were used to investigate the time course of formation and breakdown of inositol phosphates in response to receptor-active agents. In cells preincubated with [3H]inositol and in the presence of 10 mM LiCl (which blocks hydrolysis of inositol phosphate), methacholine (10(-4)M) caused a substantial increase in cellular content of [3H]inositol phosphate, [3H]inositol bisphosphate and [3H]inositol trisphosphate. Subsequent addition of atropine (10(-4) M) caused breakdown of [3H]inositol trisphosphate and [3H]inositol bisphosphate and little change in accumulated [3H]inositol phosphate. The data could be fit to a model whereby inositol trisphosphate and inositol bisphosphate are formed from phosphodiesteratic breakdown of phosphatidylinositol bisphosphate and phosphatidylinositol phosphate respectively, and inositol phosphate is formed from hydrolysis of inositol bisphosphate rather than from phosphatidyl-inositol. Consistent with this model was the finding that [3H]inositol trisphosphate and [3H]inositol bisphosphate levels were substantially increased in 5 sec while an increase in [3H]inositol phosphate was barely detectable at 60 sec. These results indicate that in the parotid gland the phosphoinositide cycle is activated primarily by phosphodiesteratic breakdown of the polyphosphoinositides rather than phosphatidyl-inositol. Also, the results show that formation of inositol trisphosphate is probably sufficiently rapid for it to act as a second messenger signalling internal Ca2+ release in this tissue.     

Polyphosphoinositide hydrolysis in endothelial cells and carotid artery segments. Bradykinin-2 receptor stimulation is calcium-independent

Bovine aortic and cerebral microvascular endothelial cells and cultured segments of canine common carotid artery possess functional receptors for the nonapeptide bradykinin which mediate a rapid increase in the formation of [3H]inositol 1-phosphate, [3H]inositol 1,4-bisphosphate, and [3H]inositol 1,4,5-trisphosphate from cell membranes containing isotopically labeled myo-inositol. Bradykinin stimulated the formation of [3H]inositol phosphates from cells in culture or tissues at threshold concentrations of 0.1 nM and 1 nM, and with a half-maximal effective concentration of 0.6-1.0 nM and 30 nM, respectively. In cultured cells, the formation of [3H]inositol trisphosphate and [3H]inositol bisphosphate preceded the formation of [3H]inositol monophosphate. Similarly, [3H]inositol phosphate formation was not inhibited by addition of calcium channel blockers, a calcium chelator, or an intracellular calcium antagonist. Calcium ionophore A23187 did not promote [3H]inositol phosphate accumulation. The receptor selectivity of the bradykinin response in cultured cells was most compatible with a type-2 mediated response. Kallidin stimulated with the same potency as bradykinin but was more potent than methionyl-lysyl-bradykinin or des-Arg9-bradykinin. The B1 receptor antagonists des-Arg9-[Leu8]-bradykinin and des-Arg10-[Leu9]-kallidin were without effect. The rapidity of the inositol phosphate response as well as the close correspondence between the bradykinin type-2 receptor mediated hydrolysis of polyphosphoinositides and changes in prostacyclin synthesis, vessel dilation, and permeability suggests that breakdown products of inositol lipids serve as second messengers mediating the effects of bradykinin on the vascular endothelium.     

The Rat Neurotensin Receptor Expressed in Chinese Hamster Ovary Cells Mediates the Release of Inositol Phosphates

To study second messenger synthesis mediated by the cloned rat neurotensin receptor, we derived a cell line stably expressing this receptor. The cDNA clone of this receptor was subcloned into the pcDNA1neo expression vector. This construct was then used to transfect Chinese hamster ovary (CHO)-K1 cells. Colony clones, selected for resistance to antibiotic G-418 sulfate, were isolated and grown separately. Nineteen individual clones were screened for total [3H]neurotensin binding as an indication of neurotensin receptor expression. The clone (CHO-rNTR-10) showing the highest level of specific [3H]neurotensin binding was characterized further. With intact cells, the equilibrium dissociation constant (KD) for specific [3H]neurotensin binding was 18 nM, and the maximal number of binding sites (Bmax) was 900 fmol/mg of protein or 740 fmol/10(6) cells (approximately 4.4 x 10(5) sites on the cellular surface). Whereas the KD was similar to that found in other cellular systems, for example, the murine neuroblastoma clone N1E-115, the Bmax exceeded previously reported values. Incubation of intact CHO-rNTR-10 cells with neurotensin caused the release of inositol phosphates in a dose-dependent manner (EC50 = 3 nM), results indicating that the expressed transfected receptor was functional. Neurotensin did not inhibit cyclic AMP levels stimulated by forskolin. As with other systems, neurotensin (8-13) was more potent than neurotensin Neurotensin-mediated inositol phosphate release is the first report of second messenger synthesis for this receptor expressed in a transfected cell line. These results suggest that the relation between structure and function of the neurotensin receptor can be readily studied in transfected cell lines.     

Stimulation of phosphatidylinositol metabolism in atrial and ventricular myocytes

Receptor-stimulated hydrolysis of inositol phospholipids was studied in atrial and ventricular myocytes isolated from guinea-pigs. Membrane phospholipids were labelled with [3H] inositol and their conversion to [3H] labelled inositol phosphate was measured in the presence of Li+ (10 mM). In the absence of added stimulatory hormones or neurotransmitters, little inositol phosphate accumulation was observed. Acetylcholine and carbachol stimulated inositol phosphate accumulation with a maximum of more than 12 times the unstimulated values in atrial myocytes and 7 times in ventricular myocytes. The EC50 values and 95% confidence limits for acetylcholine and carbachol were 0.9 microM (0.2 - 5.3) and 8.8 microM (6.3 - 11.8) in atria and 0.6 M (0.5 - 0.8) and 10.0 M (1.8 - 55.9) in ventricles, respectively. Oxotremorine was a partial agonist in stimulating inositol phosphate accumulation in both atrial and ventricular myocytes. The vasoactive peptides angiotensin II and vasopressin also stimulated inositol phosphate accumulation but the maximum effect was lower than that mediated through muscarinic receptors. However, the adenosine analogues, L-N6-phenylisopropyladenosine and 5'N-ethylcarboxamidoadenosine which, like muscarinic agonists depress cardiac contractility, did not affect inositol phosphate accumulation at concentrations up to 10(-4)M.     

Substance P-Induced Reduction in the Initial Accumulation of Cytosolic myo-[3H]Inositol in Rat Parotid Acinar Cells Mediated by the NK1 Tachykinin Receptor

Abstract: Stimulation of rat parotid acinar cells by the tachykinin neurokinin (NK) 1 receptor agonist substance P (SP) resulted in a significant reduction in the initial accumulation of cytosolic myo-[³H]inositol. This effect was rapid, because a reduction of ～15% could be seen already at 30 s, with the maximal effect (～45%) being observed at 15 min. The response to SP stimulation Was temperature dependent, because at 4°C no reduction was found, jln addition, at 4°C, cytosolic myo-[³H]inositol represented only 10% of the labeled inositol accumulated at 37°C. The SP-induced reduct on in cytosolic ravo[³H]inositol accumulation was concentration dependent; the EC₅₀ obtained for SP was 5.8 ± 2.5 nM. Spantide [N Arg¹, D-Trp⁷⁹, Leu]SP), a SP antagonist, used at a concentration oif 10⁵ A/, gave a competitive shift of the dose-response curve to SP. Various tachykinins and their analogs were evaluated for their ability to reduce cytosolic mvo-[³H]inositol. [L-Pro⁹]SP and SP methyl ester, two highly selective agonists of NK1 receptors, reduced the initial accumulation of myo-H]inositol with EQo values of 2.3 and 67.0 nM, respectively. Long SP C-terminal fragments were more potent than shorter ones. SP N-terminal fragments and SP free acid were -without effect. [Pro⁷]NKB, a selective NKB analog, had no effect. The rank order of potency of mammalian tachykinins was SP > NKA > NKB. These findings and the close correlation between EC50 values and IC₅₀ values obtained in binding studies implicate the NK 1 receptor. In addition, stimulation of muscarinic receptors by carbachol alscp resulted in a reduction in level of cytosolic mjw-[³H]inositol, with this effect being reversed by atropine. Moreover, atropine was unable tjo alter the SP-induced reduction in cytosolic myo-[³H]inositol accumulation. Other neurotransmitters, such as glutamic acid, serotonin, chplecystokinin, neurotensin, bradykinin, and neuropeptide Y, were without effect on initial cytosolic myo-[³H]inositol accumulation. In conclusion, NK1 and muscarinic receptors seem to regulate the membrane transport of inositol in acinar cells of the rat parotid gland. Measurement of the initial accumulation of cytosolic myo-[³H]inositol in this tissue could profitably be adopted as a very simple, rapid, [sensitive, and specific biochemical procedure for screening the activity of potential agonists and antagonists at NK1 receptors.     

Manganese stimulates incorporation of [3H]inositol into an agonist-insensitive pool of phosphatidylinositol in brain membranes

In a particulate preparation from rat brain, manganese ions stimulate the incorporation of [3H]inositol into inositol phospholipids in a concentration-dependent manner. Incubation with CDP-diacylglycerol (0.5 mM) alone had no effect on the incorporation of [3H]inositol but potentiated the stimulatory effect of manganese. Despite the increase in [3H]inositol incorporation into phosphatidylinositol, the carbachol-induced accumulation of [3H]inositol-1-phosphate was unaltered in membranes preincubated with manganese but when coincubated with CDP-diacylglycerol the carbachol-induced accumulation of [3H]inositol-1-phosphate was increased. These data suggest that manganese stimulates the incorporation of [3H]inositol into an agonist-insensitive pool of phosphatidylinositol.     

Effects of insulin on inositol phosphate production in cultured rat hepatocytes

Addition of vasopressin (100 nM) to rat hepatocytes prelabelled with [3H]inositol stimulated the production of inositol phosphates in the presence of 20 mM Li+. Preincubation of hepatocytes with insulin (50 nM) or glucagon (10 nM) had no significant effect alone but enhanced the effects of vasopressin after a lag period of at least 1 min. The effects of insulin and glucagon appeared additive in this respect. Insulin also enhanced the norepinephrine-mediated stimulation of inositol phosphate accumulation. The enhancement by insulin of the effects of vasopressin required at least 0.5-5 nM insulin and did not involve changes in [3H]inositol lipid labelling or IP3 phosphatase activity. The effect of insulin appeared insensitive to prior treatment of hepatocytes with pertussis toxin (200 ng/ml for 18-24 h) or cholera toxin (100 ng/ml for 3-4 h). The glucagon enhancement of the effects of vasopressin was not affected by pertussis toxin but was mimicked by cholera toxin. The response of hepatocytes to vasopressin in the absence of Li+ was smaller and more transient. Under these conditions a 5 min prior incubation with insulin inhibited the stimulation by vasopressin of inositol phosphate accumulation. A similar inhibitory effect of prior insulin exposure on the transient activation by vasopressin of exogenous phosphatidylinositol 4,5-bisphosphate breakdown by hepatocyte homogenates was also seen. These data indicate that insulin, although having no effect on basal inositol phosphate accumulation, can either enhance or antagonise the effects of vasopressin in primary rat liver hepatocyte cultures depending on the experimental conditions.     

Differential effect of progesterone on the labeling of phosphatidylinositol with [3H]inositol and [32P]phosphate in the uterus of the estrogen-treated ovariectomized rat

In rat uterine mince incubated in vitro [3H]inositol was found to be incorporated into phosphatidylinositol (PI) predominantly via a pathway which could be markedly and dose dependently activated with Mn2+ (0.1-10 mM) and inhibited by Ca2+ (1-10 mM). These ions had no effect on the incorporation of [32P]phosphate (32P) into PI indicating a distinct inositol-exchange mechanism for the labeling of PI with [3H]inositol. Treatment of ovariectomized rats for 5 days with 2 micrograms estradiol dipropionate (EDP) increased about 3-fold (when measured in the presence of 1 mM Mn2+) and 4-5-fold (when measured in the presence of 1 mM Ca2+) the inositol-exchange activity in the rat uterus, and these effects were suppressed by 40 and 30% respectively by the concomitant administration of 2 mg progesterone (P). EDP alone or in combination with P increased to the same extent (by a factor of 2-3) the rate of labeling with 32P of phosphatidylcholine (PC), phosphatidylethanolamine (PE) and plasmenylethanolamine (PmE). The labeling rate of PI was increased 1.5-1.7-fold by treatment with EDP and this increase was selectively augmented further to about 2.5-fold by the simultaneous administration of P. Treatment with P alone had no significant effect on the incorporation of either labeled precursor. Steroid hormone treatments had no effect on the amount of these phospholipids in 100 mg uterine tissue, but they increased about 1.7-fold the rate of labeling of ATP with 32P. We conclude that P, when administered together with estradiol, regulates differentially the turnover of the inositol and phosphate moieties of PI with possible physiological consequences.     

Differential sensitivity of agonist- and antagonist-occupied gonadotropin-releasing hormone receptors to protein kinase C activators. A marker for receptor activation

Gonadotropin-releasing hormone (GnRH) stimulates release of pituitary gonadotropins by activating specific plasma membrane receptors. In the present studies, we have used activators of the Ca2+- and phospholipid-dependent protein kinase (protein kinase C) to probe the binding characteristics of agonist- or antagonist-occupied GnRH receptors in intact cell cultures, using a radioligand receptor assay. Specific binding of [125I-Tyr5,D-Ser(tBu)6,Pro9,NHEt]GnRH (Buserelin), a high-affinity GnRH agonist, was increased to 180% of control in the presence of 150 nM phorbol 12-myristate 13-acetate (PMA) or 100 nM phorbol 12,13-dibutyrate (PDB), and to 125% of control in the presence of 200 microM 1,2-dioctanoylglycerol, after 20 min at 23 degrees C. The PMA effects were associated with apparent increases in both binding affinity and number of binding sites. The effects of protein kinase C activators on Buserelin binding were concentration- and time-dependent and were not seen with 4 alpha-PMA or 1,2-dioctanoyl-3-Cl-glycerol, neither of which activate protein kinase C. In contrast, PMA had no measurable effects on specific binding of a GnRH receptor antagonist, Ac[D-pCl-Phe1,2,D-Trp3,125I-Tyr5,D-Lys6,D-Ala10]GnRH. When cell cultures were pretreated with 100 nM PDB in the absence of GnRH and then washed to remove the phorbol ester, no effects of prior protein kinase C activation were detected upon subsequent addition of Buserelin. However, when PDB pretreatment was carried out in the presence of 0.3 microM GnRH, residual enhancement of Buserelin binding, but not antagonist binding, was observed at either 23 or 4 degrees C. The radiolabeled agonist activated, and the antagonist blocked, GnRH receptor-mediated luteinizing hormone release and [3H]inositol phosphate production in cells preloaded with [3H]inositol. These findings suggest that the action of protein kinase C on the GnRH receptor, either direct or indirect, requires the receptor to be in an activated (agonist-occupied) state but does not require receptor internalization. The mechanism of these effects on GnRH agonist binding is not known but may involve sequestration of surface receptors, expression of new receptors, and/or modulation of GnRH receptor affinity.