Inhibition Of Inositol Phosphate Production Research Articles

Angiotensin II type 1 receptor-function affected by mutations in cytoplasmic loop CD

To explore peptide hormone-induced conformational changes, we attempted to engineer a metal–ion binding site between the cytoplasmic loops CD and EF in the angiotensin II type 1 (AT 1) receptor. We constructed 12 double and six triple histidine mutant receptors, and tested the ability of each mutant and the wild-type to activate inositol phosphate (IP) production with and without ZnCl 2. Inhibition by ZnCl 2 in the double and triple His mutant receptors was not significant, but these mutations directly decreased the IP production. Systematic analysis of single His mutants demonstrated that the loop CD-mutants displayed 52–74% inhibition of IP production, whereas the loop EF-mutants did not affect IP production. These results indicate that the cytoplasmic loop CD-segment from Tyr 127 to Ile 130 is important for G q/11 activation by the AT 1 receptor.

Assessment of the role of G proteins and inositol phosphate production in the action of gonadotropin-releasing hormone

The first step in gonadotropin-releasing hormone (GnRH) action involves the binding of GnRH to a plasma membrane receptor. Calcium has been implicated as a second messenger molecule. More recently, it has been suggested that the products of phosphoinositide metabolism may act as a second messenger for GnRH-stimulated release of luteinizing hormone (LH). To be considered a second messenger, however, a candidate molecule must meet three requirements: in the second messenger's presence, (a) GnRH should stimulate increased production of inositol phosphate; (b) inositol phosphate production, stimulated by any means, should provoke LH release; and (c) inhibition of inositol phosphate production should block GnRH-stimulated release of LH.

Calcitonin Decreases Thyrotropin-Releasing Hormone-Stimulated Prolactin Release through a Mechanism That Involves Inhibition of Inositol Phosphate Production*

Calcitonin is present in both the hypothalamus and pituitary of the rat, and normal rat anterior pituitary cells express calcitonin receptors. Calcitonin has been reported to inhibit or to stimulate PRL release from rat anterior pituitary cells. We have investigated the effects of salmon calcitonin on basal and stimulated PRL release from rat anterior pituitary cells and have studied the effects of this peptide on the intracellular biochemical pathways involved in PRL release. Salmon calcitonin had no significant effect on basal PRL release, but inhibited (P less than 0.01) TRH-stimulated PRL release without affecting PRL release promoted by angiotensin II, neurotensin, phorbol myristate acetate (a protein kinase C activator), or maitotoxin (a calcium channel activator). Salmon calcitonin had no effect on the increase in PRL release and intracellular cAMP concentration after exposure of pituitary cells to vasoactive intestinal peptide or forskolin. Salmon calcitonin significantly decreased (P less than 0.01) the TRH-stimulated rise in inositol phosphates without affecting the angiotensin II-stimulated increase in inositol phosphates. Similarly, salmon calcitonin decreased the TRH-stimulated increase in cytosolic calcium and arachidonate liberation by pituitary cells. We conclude that salmon calcitonin selectively decreases TRH-stimulated PRL release by a mechanism that involves a decrease in inositol phosphate production, as well as a subsequent reduction in cytosolic calcium levels and in arachidonate liberation.

Pathway of phospholipase C activation initiated with platelet-derived growth factor is different from that initiated with vasopressin and bombesin.

The mode of phospholipase C activation initiated with platelet-derived growth factor (PDGF) has been studied in comparison with that initiated with vasopressin and bombesin in a rat fibroblast line, WFB. Stimulation of WFB cells by PDGF, vasopressin, and bombesin elicites rapid hydrolysis of polyphosphoinositides and an increase in cytoplasmic free Ca2+ concentration ([Ca2+]i). On stimulation by PDGF, there was a lag period of about 10 s before an increase in [Ca2+]i. No measurable lag period was observed in the [Ca2+]i response induced by vasopressin or bombesin. Pretreatment of WFB cells with phorbol 12-myristate 13-acetate profoundly inhibited inositol phosphate formation evoked by vasopressin and bombesin, but enhanced to some extent inositol phosphate formation stimulated by PDGF. In membranes prepared from WFB cells, GTP markedly augmented inositol polyphosphate formation induced by vasopressin and bombesin. It was not successful in showing the PDGF-stimulated formation of inositol phosphates in the membrane preparation. The effects of GTP, guanosine 5'-O-(2-thiodiphosphate) (GDP beta S), and guanosine 5'-O-(3-thiotriphosphate) (GTP gamma S) on polyphosphoinositide hydrolysis stimulated by growth factors were studied in WFB cells made permeable to nucleotides by treatment with either saponin or Pseudomonas aeruginosa cytotoxin. PDGF, vasopressin, and bombesin elicited inositol phosphate production in the permeabilized WFB cells in the absence of added GTP. GDP beta S, a competitive inhibitor of GTP-binding proteins (G-proteins), markedly reduced the bombesin- and vasopressin-stimulated production of inositol phosphates. However, the PDGF-stimulated production of inositol phosphates was not affected by the addition of GDP beta S. GTP gamma S, an agonist of G-proteins, largely enhanced the vasopressin- and bombesin-stimulated hydrolysis of inositol lipids when added at 10-100 microM. In the presence of GTP gamma S, the PDGF-stimulated hydrolysis of inositol lipids was not enhanced, but was reduced: 100 microM GTP gamma S reduced the stimulated hydrolysis to about a half of the control level. Only GTP gamma S, and no other nucleoside triphosphates, was found to have these effects. Activation of G-proteins in WFB cells by fluoroaluminate resulted in the inhibition of inositol phosphate production elicited with not only PDGF, but also with vasopressin and bombesin. These results indicate that a G-protein couples vasopressin and bombesin receptors to the activation of phospholipase C. Moreover, these results suggest that coupling of the PDGF receptor to phospholipase C is not mediated through a G-protein.(ABSTRACT TRUNCATED AT 400 WORDS)

Inhibition of inositol phosphate production is a late, Ca2+-dependent effect of D2 dopaminergic receptor activation in rat lactotroph cells.

The effect of dopamine, working through the activation of D2 receptors, on inositol phosphate production induced by thyrotropin-releasing hormone (TRH) was investigated in rat pituitary lactotroph cells. Dopamine (10 microM) did not modify the initial rapid stimulation of inositol 1,4,5-triphosphate and inositol bisphosphate observed within the first 15 s after TRH addition, but progressively inhibited the later inositol phosphate production induced by the neurohormone. This kinetics of inhibition was independent of dopamine preincubation time (from 2 to 10 min). The effect was still visible when dopamine was added after TRH. It was sensitive to pertussis toxin, was unchanged by increasing cellular cAMP levels with 8-Br-cAMP, but was greatly affected by treatments that modify the cytosolic free Ca2+ concentration. Specifically, the dopamine-induced inhibition was prevented by treatment of the cells with the Ca2+ ionophore ionomycin (100-200 nM) and was mimicked either by withdrawal of Ca2+ from the incubation medium or by blockade of voltage-gated Ca2+ channels with verapamil. The dopamine treatment did not decrease the cellular levels of the various phosphoinositides, strongly suggesting that the inhibition of inositol phosphate production is not due to precursor depletion. In isolated membranes, however, dopamine was unable to counteract the inositol phosphate accumulation triggered by TRH. Taken together, the data indicate that inhibition of inositol phosphate production is not a primary event triggered by D2 receptor activation, but is a late consequence, due to the previously demonstrated (Malgaroli, A., Vallar, L., Reza Elahi, F., Pozzan, T., Spada, A., and Meldolesi, J. (1987) J. Biol. Chem. 262, 13920-13927) inhibition by dopamine of the prolonged cytosolic free Ca2+ concentration increase induced by TRH via the activation of voltage-gated Ca2+ channels. These results are inconsistent with the possibility of a direct inhibitory coupling of D2 receptors to phospholipase C in rat pituitary lactotroph cells.

Dopamine D 2 receptor stimulation inhibits inositol phosphate generating system in rat striatal slices

Previous studies on the transduction mechanisms triggered by dopamine receptor stimulation have established that both D 1 and D 2 subtypes of dopamine receptors are linked to the adenylate cyclase system, the former in a stimulatory and the latter in an inhibitory manner. The present report provides the first evidence that stimulation of D 2 receptors in rat brain tissue affects the turnover of polyphosphoinositides, a as revealed by changes of the content of inositol phosphates. We found that the basal level of [ 3H]inositol trisphosphate, [ 3H]inositol bisphosphate and [ 3H]inositol monophosphate decreased following the stimulation of the D 2 receptor. The rank order of potency was quinpirole (IC 50 5 nM) > lisuride (IC 50 8 nM) > RU 24213 (IC 50 50 nM) > dopamine (IC 50 200 nM). In contrast, selective D 1 receptor stimulation by fenoldopam did not alter the inositol monophosphate, inositol bisphosphate and inositol trisphosphate content. The quinpirole effect was prevented by selective D 2 antagonists, such as domperidone and l-sulpiride (both 5 μM) while it was unaffected by the selective D 1 antagonist SCH 23390 (100 nM) and by the pharmacologically inactive d-isomer of sulpiride. Our data indicate that the activation of striatal D 2 receptors leads to the inhibition of inositol phosphate production.

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.