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PubMed1987

Adrenergic regulation of formation of inositol phosphates in rat submandibular acini.

Formation of inositol phosphates in response to adrenergic secretagogues was studied in rat submandibular acini labelled with myo-[2-3H]inositol. Noradrenaline rapidly (within 5 s) increased radioactivity incorporated into inositol 1,4,5-trisphosphate and inositol tetrakisphosphate, with less rapid (within 1 min) increases in inositol 1,3,4-trisphosphate being observed. Inositol polyphosphate formation was less sensitive to noradrenaline than was stimulation of mucin secretion and was mediated by stimulation of alpha- but not beta-adrenergic receptors. The beta-adrenergic agonist isoproterenol, which is a potent stimulator of mucin secretion [McPherson & Dormer (1984) Biochem. J. 224, 473-481] did not increase formation of inositol mono-, bis- or polyphosphates during a 15 min incubation. The results suggest that inositol phosphates do not mediate beta-adrenergic stimulation of mucin secretion in rat submandibular acini. In addition, rat submandibular acinar cells contain a Ca2+ pool which can be mobilized by isoproterenol [McPherson & Dormer (1984) Biochem. J. 224, 473-481], without involvement of inositol polyphosphates as second messengers.

Animals↗
PubMed1996

Pervanadate mediated an increased generation of inositol phosphates and tension in rat myometrium. Activation and phosphorylation of phospholipase C-gamma 1.

Stimulation of [3H]inositol-labeled rat myometrial strips with pervanadate, formed by mixing orthovanadate and H2O2, induced a dose-dependent accumulation of [3H]inositol phosphates. Orthovanadate or H2O2 added alone had no effect. Pretreatment of myometrium with two tyrosine kinase inhibitors, namely genistein and tyrphostin 47 (at 100 microM), reduced pervanadate-stimulated inositol phosphate formation by 50%. Pervanadate induced a time-sequential formation of inositol phosphates in the order inositol trisphosphate, inositol bisphosphate, and inositol monophosphate. The inhibitory effect of genistein was observed at the level of the three inositol phosphates. Pervanadate induced contraction of the myometrium; the response was dose-dependent. H2O2 or orthovanadate was without effect. Pervanadate-mediated contraction was inhibited (50%) by genistein and tyrphostin 47 (100 microM). Western blot analysis, using anti-phosphotyrosine antibodies, revealed that phosphorylated proteins were present in detergent extracts from pervanadate-stimulated myometrium. Tyrosine phosphorylation was reduced by a preincubation with 100 microM genistein or tyrphostin 47. Phospholipase C-gamma1 was immunodetected in myometrial extracts and was identified as one of the substrates subject to tyrosine phosphorylation following pervanadate treatment. The results demonstrate that, in myometrium, protein tyrosine kinase/phosphatase activities controlled both phosphorylation and activation of phospholipase C-gamma1, contributing to the modulation of the generation of inositol phosphates and tension.

Animals↗
PubMed1985

5-Hydroxytryptamine stimulates inositol phosphate production in a cell-free system from blowfly salivary glands. Evidence for a role of GTP in coupling receptor activation to phosphoinositide breakdown.

Phosphoinositide breakdown has been linked to the receptor mechanism involved in the elevation of cytosolic Ca2+. In a cell-free system prepared from [3H] inositol-labeled blowfly salivary glands, 5-hydroxytryptamine stimulated the rapid production of inositol phosphates. Within 30 s of hormone addition, there was a 100% increase in inositol trisphosphate formation, a 70% increase in inositol bisphosphate formation, and a 90% increase in inositol monophosphate formation as compared to control homogenates incubated for the same length of time. 5-Hydroxytryptamine did not stimulate inositol or glycerol phosphoinositol formation. Half-maximal activation of inositol phosphate production was obtained with 0.33 microM 5-hydroxytryptamine. Ethylene glycol bis(beta-aminoethyl ether)-N',N',N',N'-tetraacetic acid, (EGTA) (0.3 mM) inhibited the basal formation of inositol phosphates and decreased the net accumulation of inositol bisphosphate and inositol trisphosphate due to hormone as compared to homogenates incubated in the absence of added Ca2+. EGTA, however, had little effect on the per cent stimulation of inositol phosphate production due to hormone. In homogenates, ATP, GTP or guanyl-5'-yl imidodiphosphate (Gpp(NH)p) was required for a hormone effect. Gpp(NH)p, unlike ATP or GTP, increased the basal formation of inositol phosphates. In membranes, GTP, Gpp(NH)p, or guanosine 5'-(3-O-thio)trisphosphate (GTP gamma S) sustained a hormone effect whereas ATP was ineffective. GTP did not affect production while Gpp(NH)p and GTP gamma S increased inositol phosphate production. Half-maximal effects of Gpp(NH)p and GTP gamma S on hormone-stimulated inositol phosphate formation occurred at 10 microM and 100 nM, respectively. In the presence of 1 microM GTP gamma S, 5-methyltryptamine stimulated inositol phosphate formation within 2 s in membranes. These results indicate that in a cell-free system, GTP is involved in mediating the effects of Ca2+-mobilizing hormones on phosphoinositide breakdown.

Animals↗
PubMed1996

TGF-beta isoforms fail to modulate inositol phosphates and cAMP in normal and tumour-derived human oral keratinocytes.

This study examined inositol phosphate and cAMP regulation by TGF-beta 1, -beta 2 and -beta 3 in normal and tumour-derived human oral keratinocytes. Previous findings indicated that the cell lines expressed TGF-beta cell surface receptors and had a range of response to exogenous TGF-beta 1, -beta 2 and -beta 3 from being refractory to the ligand to marked inhibition. Basal levels of inositol phosphates broadly reflected the differentiation status of the cells as demonstrated by involucrin expression, but did not correlate with responsiveness to TGF-beta 1, as measured previously by thymidine incorporation. Treatment of cells with bradykinin or serum caused up-regulation of inositol phosphate levels; by contrast, TGF-beta 1, -beta 2 and -beta 3 failed to modulate inositol phosphates. In two tumour-derived cell lines, the TGF-beta isoforms had no effect on cAMP levels, despite a significant increase in cAMP using a potent agonist of adenylate cyclase (forskolin). Furthermore, the cAMP analogue, dibutyryl cAMP, failed to mimic the inhibitory or refractory responses of TGF-beta in these cells lines. The results demonstrate that in normal and tumour-derived human oral keratinocytes, TGF-beta signal transduction is not mediated by inositol phosphates or cAMP.

Bradykinin↗
PubMed1997

HIV-1 envelope protein, gp120, has no effects on inositol phosphate production and metabolism in the Jurkat T-cell line either in the presence or absence of receptor stimulation.

We have used HPLC techniques to investigate the effects of gp120 upon inositol phosphate turnover in Jurkat E6-1 CD4+ T-cells, to pursue previous reports that this viral coat protein: (a) inhibits receptor-activated inositol phosphate release; (b) stimulates basal inositol phosphate release; (c) inhibits inositol polyphosphate 5-phosphatase. Treatment of cells with up to 10 microg/ml gp120 from between 10 min and 24 h was without effect upon inositol phosphate turnover in both basal cells, and in C305 and OKT3 stimulated cells. This is the first report that biologically competent gp120 does not affect any aspect of inositol phosphate turnover in either basal or receptor-activated lymphocytes.

Chromatography, High Pressure Liquid↗
PubMed1992

Inhibitory effects of isolated inositol phosphates on zinc absorption in humans.

Inositol hexa phosphate (phytic acid) forms poorly soluble complexes with zinc, resulting in reduced absorption. During food preparation and processing phytate is partly hydrolyzed to lower inositol phosphates which have other binding properties. Zinc absorption in humans was measured in 18 women (22-37 a) by use of extrinsic labelling of meals with 65Zn and measurement of whole body retention. Inositol phosphates--400 mumol of the hexa phosphate form (IP-6), 200 and 400 mumol of the penta phosphate form (IP-5) and 400 mumol of the tetra phosphate form (IP-4)--were added to white bread and served with 200 mL of milk. The zinc content of the meal was 22 mumol. The absorption of zinc (65Zn) from white bread with no detectable phytate content was 43.3 +/- 17.9% (mean +/- SD) and from the breads with added 400 mumol of IP-6: 14.3 +/- 3.2%; 200 mumol of IP-5: 27.1 +/- 5.3%; 400 mumol of IP-5: 18.1 +/- 4.2%; and 400 mumol of IP-4: 41.5 +/- 11.3%. The results indicate that IP-6 and IP-5 have zinc-absorption depressing properties, while IP-4 added in isolated form is relatively inert as regards zinc absorption. However, a comparison with earlier obtained results from zinc absorption studies indicate that results obtained with isolated inositol phosphates can not directly be extrapolated to the phytate contents of foods.

Absorption↗
PubMed1998

Diphospho-myo-inositol phosphates in Dictyostelium and Polysphondylium: identification of a new bisdiphospho-myo-inositol tetrakisphosphate.

The two major diphospho inositol phosphates from the axenic strain Dictyostelium discoideum AX2 were previously investigated and identified as 6-PP-InsP5 and 5,6-bis-PP-InsP4. In order to examine whether these findings are representative of Dictyostelids in general, five non-axenic wild-type species of Dictyostelium and two of Polysphondylium were studied. It was found that all of the Dictyostelium species exhibit similar patterns of diphospho inositol phosphates. By contrast, both of the Polysphondylium species contain 5-PP-InsP5 as the predominant isomer. Besides 5,6-bis-PP-InsP4, a new bis-PP-InsP4 was detected in Polysphondylium. This compound is either 1,5-bis-PP-InsP4 or its corresponding enantiomer 3,5-bis-PP-InsP5. The structures were elucidated by two-dimensional 1H-1H and 1H-31P NMR analysis. Additionally, they were confirmed using a specific 6-PP-InsP(5)-5-kinase from D. discoideum AX2 as an enantio-specific tool and enantiomerically pure reference standards.

Animals↗
PubMed1988

The separation of myo-inositol phosphates by ion-pair chromatography.

The separation of myo-inositol phosphates by ion pair, reverse-phase high performance liquid chromatography has been investigated. The retention of the inositol phosphates is dependent on both the polarity of the hetaeron utilized and on the pH of the solvent. A method is presented which permits the isocratic separation of multiple forms of inositol phosphates including isomers of myo-inositol trisphosphate. This method appears to be superior to the anion exchange based systems currently employed because of smaller retention volumes, the low ionic strength of the solvent employed, the absence of a requirement for reequilibration, and the ability to perform separations isocratically.

Chromatography, High Pressure Liquid↗
PubMed1987

Inositol phosphate metabolism in bradykinin-stimulated human A431 carcinoma cells. Relationship to calcium signalling.

Stimulation of human A431 epidermoid carcinoma cells by bradykinin causes a very rapid release of inositol phosphates and a transient rise in cytoplasmic free Ca2+ concentration ([Ca2+]i). Bradykinin-induced inositol phosphate formation is half-maximal at a concentration of 4 nM and is not affected by pertussis toxin. H.p.l.c. analysis of the various inositol phosphates shows an immediate but transient accumulation of inositol 1,4,5-trisphosphate [Ins(1,4,5)P3], which reaches a peak value of approx. 10 times the basal level within 15 s and slightly precedes the rise in [Ca2+]i, both parameters changing in parallel. After a lag period, bradykinin also induces a massive accumulation of Ins(1,3,4)P3 and inositol 1,3,4,5-tetrakisphosphate [Ins(1,3,4,5)P4]. Our data support the view that part of the newly formed Ins(1,4,5)P3 is converted into Ins(1,3,4)P3 phosphorylation/dephosphorylation with Ins(1,3,4,5)P4 as intermediate. Furthermore, A431 cells were found to contain strikingly high basal levels of two other inositol phosphates, presumably inositol pentakisphosphate (InsP5) and inositol hexakisphosphate (InsP6), representing more than 50% of the total 3H radioactivity incorporated into inositol phosphates. The presumptive InsP5 and InsP6 are only slightly affected by bradykinin. Although Ins(1,3,4)P3 and InsP4 could function as second messengers, our results suggest that, unlike Ins(1,4,5)P3, neither Ins(1,3,4)P3 nor InsP4 are involved in Ca2+ mobilization.

Bradykinin↗
PubMed1988

Epidermal-growth-factor-induced formation of inositol phosphates in human A431 cells. Differences from the effect of bradykinin.

In human A431 epidermoid carcinoma cells, epidermal growth factor (EGF) rapidly stimulates the breakdown of inositol phospholipids and raises cytoplasmic free [Ca2+]. In this paper, we investigate the action of EGF on inositol phosphate metabolism, and we compare it with the previously described effects of bradykinin on the same cell system [Tilly, van Paridon, Verlaan, Wirtz, de Laat & Moolenaar (1987) Biochem. J. 244, 129-135]. In cells prelabelled with [3H]inositol, EGF slowly but persistently (for at least 30 min) stimulates the formation of [3H]inositol phosphates, whereas bradykinin causes an immediate but transient release of inositol phosphates, which lasts for only a few minutes. The EGF effect is additive to bradykinin stimulation and does not require extracellular Ca2+. In contrast, inositol phosphate formation induced by Ca2+-ionophore A23187 has an absolute requirement for external Ca2+. Treatment of the cells with 12-O-tetradecanoylphorbol 13-acetate completely abolishes the response to EGF and to sub-optimal doses of bradykinin, suggesting a negative-feedback function of protein kinase C. Pretreatment of the cells with pertussis toxin has no effect on inositol phosphate formation induced by either EGF or bradykinin. Unlike bradykinin, EGF stimulates very little accumulation of inositol 1,4,5-trisphosphate, with only a small and rather variable release of Ca2+ from intracellular stores. EGF rapidly but transiently increases inositol 1,3,4-trisphosphate and 1,3,4,5-tetrakisphosphate, but the effects are much smaller than those of bradykinin. In addition, EGF increases both inositol mono- and bis-phosphate. At 10 min after EGF addition, inositol monophosphate, unlike the other inositol phosphates, is still increasing. It is concluded that the EGF-dependent pattern of stimulation is different from that observed in bradykinin-stimulated A431 cells, suggesting that there are separate mechanisms of inositol-lipid hydrolysis involved.

Bradykinin↗
PubMed1995

Substance P-related antagonists inhibit vasopressin and bombesin but not 5'-3-O-(thio)triphosphate-stimulated inositol phosphate production in Swiss 3T3 cells.

The substance P (SP) analogues [DArg1, DPhe5, DTrp7,9, Leu11] SP (AntD) and [Arg6, DTrp7,9, MePhe8] SP (6-11) (AntG) inhibit the action of many different neuropeptides including SP. These analogues might be useful in the treatment of small cell lung cancer but their mechanism of action is unclear. Here, we analyzed the effect of AntD and AntG on neuropeptide vs. guanosine 5'-3-O-(thio) triphosphate (GTP gamma S)-stimulated inositol phosphate generation in permeabilized Swiss 3T3 cells. AntD inhibited vasopressin and bombesin stimulated inositol phosphate formation (IC50 of 0.75 microM and 2 microM, respectively). Similarly, AntG inhibited vasopressin-stimulated inositol phosphate generation with an IC50 of 1 microM. Strikingly, neither AntD up to 10 microM nor AntG up to 20 microM was able to inhibit GTP gamma S-stimulated inositol phosphate generation. Dose-response curves of neuropeptide-induced inositol phosphate generation were dramatically displaced to the right by either 10 microM AntD or 20 microM AntG. However, neither antagonist affected the dose response of GTP gamma S-stimulated inositol phosphate generation. Furthermore, 20 microM AntD had no effect on AIF-4-induced inositol phosphates in COS-1 cells transfected with G alpha q. AntD inhibited [3H]vasopressin binding competitively in intact Swiss 3T3 cells and both AntD and AntG inhibited [3H]vasopressin binding in Swiss 3T3 and rat liver membranes. Scatchard analysis revealed that AntD inhibited vasopressin binding by reducing receptor affinity without affecting receptor number in both intact and membrane preparations of Swiss 3T3 cells. The results strongly suggest that SP analogues AntD and AntG block the action of the Ca2+ mobilizing neuropeptides at the receptor level, rather than inhibiting G protein-stimulated inositol phosphate production.

3T3 Cells↗
PubMed1990

Platelet activating factor induces inositol phosphate accumulation in cultures of rat and bovine anterior pituitary cells.

The phospholipid platelet-activating factor (PAF) (1-O-alkyl-2-acetyl-sn-glycero-3-phosphocholine) stimulated the accumulation of inositol phosphates in cultures of rat and bovine anterior pituitary cells. In response to PAF, inositol 1,4-bisphosphate showed the largest percent increase of the inositol phosphates in the presence of lithium chloride. PAF induced an increase of inositol 1,4,5-trisphosphate, the biologically active isomer responsible for mobilization of intracellular calcium. A characterization of the PAF response indicated that PAF, but not its biologically inactive enantiomer, induced the accumulation of inositol phosphates in the rat anterior pituitary. Further, the PAF receptor antagonist L652731 reduced PAF stimulation. The ED50 for PAF-induced inositol 1,4-bisphosphate accumulation was 0.4 nM. PAF induced a rapid response that did not persist beyond 20 min. While PAF treatment of anterior pituitary cells did not alter TRH-induced inositol phosphate accumulation, it did prevent a second exposure of PAF from inducing inositol phosphate accumulation. These data suggest that PAF induces a rapid stimulation of phospholipase C causing the hydrolysis of phosphatidylinositol 4,5-bisphosphate and the generation of the second messengers, inositol 1,4,5-trisphosphate and diglyceride, in anterior pituitary tissue. This action is transient probably due to PAF receptor desensitization. The action of PAF on generation of inositol phosphates may account, in part, for PAF-induced secretion of PRL and GH.

Animals↗
PubMed1988

Formation of inositol phosphates in synaptoneurosomes of guinea pig brain: stimulatory effects of receptor agonists, sodium channel agents and sodium and calcium ionophores.

Carbamylcholine, norepinephrine, histamine and glutamate stimulated the formation of [3H]inositol phosphates in [3H]inositol-labelled synaptoneurosomes obtained from the cortex, striatum and hippocampus of the guinea pig. Synaptoneurosomes prepared from the cerebellum did not respond to receptor agonists. Agents that enhance the influx of sodium ions through voltage-sensitive channels, such as batrachotoxin, scorpion venom and pumiliotoxin B, or a sodium ionophore, monensin, stimulated the formation of [3H]inositol phosphates in synaptoneurosomes from all four regions of the brain. Neither calcium channel blockers nor receptor antagonists reduced the responses to batrachotoxin. Ionomycin, a calcium ionophore, also stimulated the formation of [3H]inositol phosphates in synaptoneurosomes from all four regions of the brain. A phorbol ester inhibited the formation of [3H]inositol phosphates, elicited by either receptor agonists or by sodium channel agents. The major [3H]inositol-labelled lipid in the synaptoneurosomes was phosphatidylinositol as analyzed by thin layer chromatography. While the hydrolysis of phosphatidylinositol elicited by carbamylcholine resulted in an increase of lipid-labelling with [3H]inositol, sodium channel agents caused a decrease in the incorporation of [3H]inositol. The results indicate that intracellular sodium may have a regulatory role in the turnover of phosphatidylinositol, and that, unlike the receptor-mediated responses, this regulation is present in all regions of the brain.

Adenosine Triphosphate↗
PubMed1993

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.

Animals↗
PubMed2005

Interaction of the catalytic domain of inositol 1,4,5-trisphosphate 3-kinase A with inositol phosphate analogues.

The levels of inositol 1,4,5-trisphosphate [Ins(1,4,5)P3] in the cytoplasm are tightly regulated by two enzymes, Ins(1,4,5)P3 3-kinase and type I Ins(1,4,5)P3 5-phosphatase. The catalytic domain of Ins(1,4,5)P3 3-kinase (isoenzymes A, B and C) is restricted to approximately 275 amino acids at the C-terminal end. We were interested in understanding the catalytic mechanism of this key family of enzymes in order to exploit this in inhibitor design. We expressed the catalytic domain of rat Ins(1,4,5)P3 3-kinase A in Escherichia coli as a His- and S-tagged fusion protein. The purified enzyme was used in an Ins(1,4,5)P3 kinase assay to phosphorylate a series of inositol phosphate analogues with three or four phosphate groups. A synthetic route to D-2-deoxy-Ins(1,4,5)P3 was devised. D-2-Deoxy-Ins(1,4,5)P3 and D-3-deoxy-Ins(1,4,6)P3 were potent inhibitors of the enzyme, with IC50 values in the micromolar range. Amongst all analogues tested, only D-2-deoxy-Ins(1,4,5)P3 appears to be a good substrate of the Ins(1,4,5)P3 3-kinase. Therefore, the axial 2-hydroxy group of Ins(1,4,5)P3 is not involved in recognition of the substrate nor does it participate in the phosphorylation mechanism of Ins(1,4,5)P3. In contrast, the equatorial 3-hydroxy function must be present in that configuration for phosphorylation to occur. Our data indicate the importance of the 3-hydroxy function in the mechanism of inositol trisphosphate phosphorylation rather than in substrate binding.

Animals↗
PubMed1990

Decapitation-induced changes in inositol phosphates in rat brain.

Decapitation resulted in a time-dependent production of inositol phosphates in rat brain. This production was analyzed by measuring both the radioactivity and the concentrations of inositol phosphates generated from [3H]inositol-labeled phospholipids. Both measurements produced the same time-dependent changes, including a rapid decrease in inositol 1,4,5-trisphosphate within 1.5 min, a 6-fold increase in inositol 1,4-bisphosphate to a maximum at 1.5 min, a 5-fold rise in inositol 4-monophosphate to a maximum at 2.5 min, and little change in inositol 1-monophosphate. The temporal changes in the mass and radioactivity of these compounds, together with the decrease in labeling of phosphatidylinositol 4,5-bisphosphates, support the idea that the inositol phosphates originate from the hydrolysis of phosphatidylinositol 4,5-bisphosphates and not from either the direct hydrolysis of phosphatidylinositol 4-phosphates or phosphatidylinositols.

Animals↗
PubMed1993

Fatty acid inhibition of angiotensin II-stimulated inositol phosphates in smooth muscle cells.

Inositol phosphate (InsP) responses to angiotensin II (ANG II) stimulation were measured in cultured rat vascular smooth muscle cells (VSMC) incubated with and without fatty acids (FA). VSMC were washed after 24 h of FA incubation to achieve cellular incorporation of FA yet eliminate ambient FA. Incubation with eicosapentaenoic acid (EPA)-supplemented medium resulted in concentration-dependent incorporation of EPA and depletion of arachidonic acid in VSMC membranes. Incubation with EPA, but not other FA, resulted in inhibition of ANG II-stimulated InsP formation (29% inhibition with 100 microM EPA). In contrast, InsP formation in response to guanine nucleotide-binding protein stimulation was not affected by EPA. ANG II receptor binding to membranes prepared from EPA-loaded VSMC was 18% lower than binding in membranes from sham-loaded cells. In other studies, VSMC were exposed acutely to FA to avoid cellular incorporation. Exposure to all FA resulted in concentration-dependent reductions in ANG II binding and ANG II-stimulated InsP formation; binding affinity was reduced without changes in receptor density. We conclude that ANG II-stimulated InsP formation is modestly and selectively inhibited by EPA incorporation and more profoundly inhibited by acute exposure to many FA via interference with ANG II receptor binding.

Angiotensin II↗
PubMed1996

Beta adrenergic receptor activation attenuates the generation of inositol phosphates in the pregnant rat myometrium. Correlation with inhibition of Ca++ influx, a cAMP-independent mechanism.

In the pregnant rat myometrium, an averaged 30% of inositol phosphate accumulation induced by carbachol and oxytocin was inhibited by oxodipine indicating that a part of receptor-mediated generation of inositol phosphates depended on Ca++ influx through voltage-gated Ca++ channels. In fura-2-loaded cells, carbachol and oxytocin caused a two-phase [Ca++]i response, made up of a transient [Ca++]i peak of about 700 nM followed by a sustained phase of about 120 nM. Oxodipine reduced the [Ca++]i peak by 40% and the plateau phase by 50%, pointing to a contribution of Ca++ influx in both the [Ca++]i peak and sustained phase. Isoproterenol reduced inositol phosphate response to carbachol and oxytocin to an amount equivalent to that elicited by oxodipine. No additional reduction could be obtained in a combination of isoproterenol and oxodipine. Isoproterenol decreased by 40% the [Ca++]i peak and by 70% the [Ca++]i plateau phase. Differently from isoproterenol, forskolin did not affect inositol phosphate accumulation induced by oxytocin and failed to attenuate the [Ca++]i peak. The inhibitory effect of isoproterenol on both inositol phosphate accumulation and [Ca++]i increase induced by oxytocin was abolished by pertussis toxin. These data suggest that beta adrenergic receptor activation is linked via a cAMP-independent, pertussis toxin-sensitive process to an activation of K+ channels, as revealed by use of selective K+ channel antagonists, with the consequent closure of voltage-gated Ca++ channels, resulting in the inhibition of the Ca(++)-associated generation of inositol phosphates.

Acetylcholine↗