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Analysis of inositol by high-performance liquid chromatography
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A high-performance liquid chromatographic (HPLC) method has been developed for identification and quantification of inositol isomers and monosaccharides in inositol-containing glycans. The method, which can determine 10 pmol of inositol, utilizes an Aminex HPX-87C column packed with an 8% crosslinked cation-exchange resin in the calcium form eluted with deionized water at 50 degrees C. NaOH solution is added to the column effluent through a postcolumn tee to increase the pH (pH greater than 11.6) before entering a pulsed amperometric detector which is highly sensitive for polyhydroxylated compounds. Samples in which inositol is linked to sugar through a glycosidic bond are hydrolyzed with 5.5 N trifluoroacetic acid, 100 degrees C, 4 h, and then reduced with NaBH4. Samples in which inositol is linked via a phosphate ester are hydrolyzed with 6 N HCl, 110 degrees C, 24 h. This method has been applied to the analysis of inositol in the hamster prion proteins (PrP) PrP27-30, and PrPSc.
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... Dionex HPLC Analysis-It has been shown previously that chiroand myoinositol (as well as other isomers) can be separated by using the Dionex HPLC System (16). In this study, samples were analyzed by the Dionex Bio LC Gradient Pump Module GPM-2 System. ...
... is the most abundant hexahydroxycyclohexane found (16). Myoinositol was first shown to be synthesized in uiuo from labeled glucose (22). ...
We report here the in vivo conversion of [3H]myoinositol to [3H]chiroinositol. After labeling intraperitoneally with [3H]myoinositol for 3 days to reach radioisotope equilibrium in urine, [3H]chiroinositol was isolated from tissues and purified after 6 N HCl hydrolysis by two sequential paper chromatographies and high performance liquid chromatography (HPLC). Percent conversion of [3H]myoinositol to [3H]chiroinositol was highest in urine (36%), liver (8.8%), muscle (8.8%), and blood (7.6%) with intestine, brain, kidney, spleen, and heart decreasing in percentage from 2.8 to 0.7%. Labeling of other inositol isomers including scyllo-, neo-, and epi-, and mucoinositol was minimal, approximately 0.06% of [3H]myoinositol. Glucose was unlabeled, but glucuronate, the product of myoinositol oxidation, was labeled up to 1.5% of the [3H] myoinositol. Acid hydrolysates of combined inositol-containing phospholipids contain significant labeled chiroinositol. [3H]Phosphatidylinositols and [3H]glycosylphosphatidylinositols were extracted from liver, muscle, and blood, isolated by thin layer chromatography, and inositols purified by HPLC after acid hydrolysis. Percent conversion of [3H]myoinositol to [3H] chiroinositol was highest in blood (60.4%) followed by muscle (7.7%) and liver (2.2%).
... This is the reason for the name "inositol"-from the Greek term "is" (genitive "inos"), which means "muscle" [13]. Only later was the cyclohexanol structure established, although the precise conformation of isomers has only been described in recent times [14]. The basic hexahydroxycyclohexane backbone allows for the establishment of nine isomers (cis-, epi-, allo-, myo-, neo-, scyllo-, L-chiro-, D-chiroand muco-inositol). ...
Myo-inositol is a natural polyol, the most abundant among the nine possible structural isomers available in living organisms. Inositol confers some distinctive traits that allow for a striking distinction between prokaryotes and eukaryotes, the basic clusters into which organisms are partitioned. Inositol cooperates in numerous biological functions where the polyol participates or by furnishing the fundamental backbone of several related derived metabolites, mostly obtained through the sequential addition of phosphate groups (inositol phosphates, phosphoinositides, and pyrophosphates). Overall myo-inositol and its phosphate metabolites display an entangled network, which is involved in the core of the biochemical processes governing critical transitions inside cells. Noticeably, experimental data have shown that myo-inositol and its most relevant epimer D-chiro-inositol are both necessary to permit a faithful transduction of insulin and of other molecular factors. This improves the complete breakdown of glucose through the citric acid cycle, especially in glucose-greedy tissues, such as the ovary. In particular, while D-chiro-inositol promotes androgen synthesis in the theca layer and down-regulates aromatase and estrogen expression in granulosa cells, myo-inositol strengthens aromatase and FSH receptor expression. Inositol effects on glucose metabolism and steroid hormone synthesis represent an intriguing area of investigation, as recent results have demonstrated that inositol-related metabolites dramatically modulate the expression of several genes. Conversely, treatments including myo-inositol and its isomers have proven to be effective in the management and symptomatic relief of a number of diseases associated with the endocrine function of the ovary, namely polycystic ovarian syndrome.
... In the middle of the 19th century, Johann Joseph Scherer extracted a polyol-a hexahydroxy-cyclohexane compound-hence named "inositol"-from muscle cells [41]. The chemical structure was ascertained later, and the configuration of all of its related isomers only recently has been clearly described [42]. Noticeably, the hexahydroxycyclohexane backbone displays a bewildering plasticity in allowing the emergence of nine different isomers: cis-, epi-, allo-, myo-, neo-, scyllo-, L-chiro-, D-Chiro-, and muco-inositol. ...
Polycystic ovarian syndrome (PCOS) is the most common endocrinological disorder in women, in which, besides chronic anovulation/oligomenorrhea and ovarian cysts, hyperandrogenism plays a critical role in a large fraction of subjects. Inositol isomers—myo-Inositol and D-Chiro-Inositol—have recently been pharmacologically effective in managing many PCOS symptoms while rescuing ovarian fertility. However, some disappointing clinical results prompted the reconsideration of their specific biological functions. Surprisingly, D-Chiro-Ins stimulates androgen synthesis and decreases the ovarian estrogen pathway; on the contrary, myo-Ins activates FSH response and aromatase activity, finally mitigating ovarian hyperandrogenism. However, when the two isomers are given in association—according to the physiological ratio of 40:1—patients could benefit from myo-Ins enhanced FSH and estrogen responsiveness, while taking advantage of the insulin-sensitizing effects displayed mostly by D-Chiro-Ins. We need not postulate insulin resistance to explain PCOS pathogenesis, given that insulin hypersensitivity is likely a shared feature of PCOS ovaries. Indeed, even in the presence of physiological insulin stimulation, the PCOS ovary synthesizes D-Chiro-Ins four times more than that measured in control theca cells. The increased D-Chiro-Ins within the ovary is detrimental in preserving steroidogenic control, and this failure can easily explain why treatment strategies based upon high D-Chiro-Ins have been recognized as poorly effective. Within this perspective, two factors emerge as major determinants in PCOS: hyperandrogenism and reduced aromatase expression. Therefore, PCOS could no longer be considered a disease only due to increased androgen synthesis without considering the contemporary downregulation of aromatase and FSH receptors. Furthermore, these findings suggest that inositols can be specifically effective only for those PCOS phenotypes featured by hyperandrogenism.
... It began in 1850 when the German physician and chemist, Johann Joseph Scherer, isolated a hexahydroxycyclohexane from muscle cells and named this molecule "Inositol" from the combination of the Greek terms [ìς (is, in-, "sinew, fiber"), -ose (indicating a carbohydrate), -ite ("ester"), -ol ("an alcohol")] and also because of its sweet taste [1]. Only many years later, Maquenne established the inositol cyclohexanol structure, purifying it from leaves [2], while a century later the elegant work of Posternak described the configuration of the main inositol isomer in eukaryotic tissues: myo-inositol (myo-Ins) [3]. The structure of this hexahydroxycyclohexane allows the formation of nine different isomers: cis-, epi, allo-, myo-, neo-, scyllo-, L-chiro-, D-chiro-and muco-inositol ( Figure 1) [4]. ...
Myo-inositol (myo-Ins) and D-chiro-inositol (D-chiro-Ins) are natural compounds involved in many biological pathways. Since the discovery of their involvement in endocrine signal transduction, myo-Ins and D-chiro-Ins supplementation has contributed to clinical approaches in ameliorating many gynecological and endocrinological diseases. Currently both myo-Ins and D-chiro-Ins are well-tolerated, effective alternative candidates to the classical insulin sensitizers, and are useful treatments in preventing and treating metabolic and reproductive disorders such as polycystic ovary syndrome (PCOS), gestational diabetes mellitus (GDM), and male fertility disturbances, like sperm abnormalities. Moreover, besides metabolic activity, myo-Ins and D-chiro-Ins deeply influence steroidogenesis, regulating the pools of androgens and estrogens, likely in opposite ways. Given the complexity of inositol-related mechanisms of action, many of their beneficial effects are still under scrutiny. Therefore, continuing research aims to discover new emerging roles and mechanisms that can allow clinicians to tailor inositol therapy and to use it in other medical areas, hitherto unexplored. The present paper outlines the established evidence on inositols and updates on recent research, namely concerning D-chiro-Ins involvement into steroidogenesis. In particular, D-chiro-Ins mediates insulin-induced testosterone biosynthesis from ovarian thecal cells and directly affects synthesis of estrogens by modulating the expression of the aromatase enzyme. Ovaries, as well as other organs and tissues, are characterized by a specific ratio of myo-Ins to D-chiro-Ins, which ensures their healthy state and proper functionality. Altered inositol ratios may account for pathological conditions, causing an imbalance in sex hormones. Such situations usually occur in association with medical conditions, such as PCOS, or as a consequence of some pharmacological treatments. Based on the physiological role of inositols and the pathological implications of altered myo-Ins to D-chiro-Ins ratios, inositol therapy may be designed with two different aims: (1) restoring the inositol physiological ratio; (2) altering the ratio in a controlled way to achieve specific effects.
... Naturally occurring isomers of inositol are myoinositol, chiro-inositol, scyllo-inositol, muco-inositol, and neo-inositol with myo-inositol being the most abundant (Wang et al., 1990). Inositols and their various isomers are not all present in all plants or animals. ...
McDonald IV, L. W., S. C. Goheen, P. A. Donald, J. A. Campbell. 2012. Identification and quantitation of various inositols and o-methylinositols present in plant roots related to soybean cyst nematode host status. Nematropica 42:1-8. Inositols and O-methylinositols in plant roots were extracted, isolated, and analyzed to test the hypothesis that these compounds may be involved in feeding site establishment for soybean cyst nematode. Root samples from soybean (Glycine max), tapia bean (Phaseolus vulgaris), crimson clover (Trifolium incarnatum), corn (Zea mays), sugar beet (Beta vulgaris), and peanut (Arachis hypogaea) were extracted with 80% ethanol, derivatized, and analyzed using gas chromatography/mass spectrometry (GC/MS). Fragment ions at m/z 260 and 265 were used to quantify the O-methylinositols and inositols, respectively. Pinitol, D-chiro-inositol, and myo-inositol were found in soybean samples and myo-inositol in tapia bean. Sugar beets contained D-chiro-inositol. Pinitol and myo-inositol were found in crimson clover. Corn contained myo-inositol. Pinitol, D-chiro-inositol, ononitol, and myo-inositol were found in peanuts. No correlation was found between the presence of any of the inositols or o-methyl inositols and the ability of these plants to host the soybean cyst nematode. ResUMen McDonald IV, L. W., S. C. Goheen, P. A. Donald, J. A. Campbell. 2012. Identificación y cuantificación de varios inositoles y o-metilinositoles presentes en raíces y su relación con la susceptibilidad al nematodo quiste de la soya. Nematropica 42:1-8. Se extrajeron, aislaron y analizaron los inositoles y o-metilinositoles en raíces de plantas para probar la hipótesis de la participación de estos compuestos en el establecimiento de sitios de alimentación del nematodo quiste de la soya. Se incluyeron raíces de soya (Glycine max), fríjol (Phaseolus vulgaris), trébol rojo (Trifolium incarnatum), maíz (Zea mays), remolacha azucarera (Beta vulgaris) y maní (Arachis hypogaea) y se extrajeron las muestras con 80% etanol, se derivatizaron y se analizaron usando cromatografía de gases/espectrometría de masas (GC/MS). Se usaron fragmentaciones a m/z 260 y 265 para cuantificar los o-metilinositoles y los inositoles, respectivamente. En las muestras de soya se encontró pinitol, D-chiro-inositol y myo-inositol, y se encontró myo-inositol en el fríjol. La remolacha azucarera contuvo D-chiro-inositol. En el trébol rojo se halló Pinitol y myo-inositol. El maíz mostró myo-inositol y en el maní se encontró pinitol, D-chiro-inositol, ononitol y myo-inositol. No se halló ninguna correlación entre la presencia de ningúno de los inositoles o de los o-metilinositoles y la habilidad de estas plantas para ser hospedante del nematodo quiste de la soya. Palabras clave: Cromatografía de gases/espectrometría de masa, Inositol, O-metil-inositol, Raíces
... The analysis of free myo-inositol in foods and biological tissues has generally been facilitated following a simple protein precipitation step in advance of end-point analysis. Methods targeting the aggregate of free and bound forms have used acidic or alkaline hydrolysis followed by traditional microbiological assay (83), enzymatic assay (84), GC subsequent to previous derivatization (85)(86)(87)(88), HPLC or high-performance anion-exchange chromatography (HPAEC) techniques using UV, evaporative light-scattering detection (ELSD), or pulsed amperometric detection (PAD) (82,(89)(90)(91)(92)(93)(94), and HPLC or UHPLC coupled with MS (95)(96)(97). A review has summarized the techniques that are applicable for the analysis of inositol and related compounds, albeit specific to soybean tissues (98). ...
Infant formula is designed to provide the human infant with a sole source of nutrition and it is intended to imitate breast milk. In recent years, advances in the science of infant nutrition have led to an increasing number of novel ingredients that are supplemented into infant formula. As the list of these nutritionally important nutrients is lengthy, this review summarizes contemporary analytical methods that have been applied to a representative selection (lutein, carnitine, choline, nucleotides, inositol, taurine, sialic acid, gangliosides, triacylglycerides, oligosaccharides, α-lactalbumin, and lactoferrin).
PCOS is the commonest cause of anovulatory infertility. Many potential therapies are available and the mainstay of treatment centers around enhancing general health to achieve the best reproductive outcome for mother and baby. We describe the pathophysiology of anovulation in brief before an outline of pragmatic evidence-based algorithm for ovulation induction therapies.
Polycystic ovary syndrome (PCOS) is a common endocrine and metabolic disorder characterized by oligo-anovulation, hyperandrogenism, and insulin resistance, this latter with a key role in the pathogenesis of this syndrome. Considering the heterogeneity of this condition, various therapeutic approaches have been attempted in PCOS women, besides the classical treatments such as metformin or pioglitazone.
Emerging data support the use of natural compounds such as inositols, myo- and D-chiro-inositol, alone or in combination. Moreover, vitamins and dietary factors such as vitamin D, alpha-lipoic acid, omega 3, melatonin and probiotics may be efficient in PCOS treatment counteracting its endocrine and metabolic aspects, as well as their secondary complications.
The present chapter addresses the current knowledge about the efficacy of these nutrients in the treatment of PCOS, in view of experimental and clinical studies.
A high performance anion-exchange chromatographic method employing pulsed amperometric detection was applied to the determination of endogenous free and total myo-inositol in bovine milk, with the objective of characterising the distribution between its free and bound forms, about which little is known. The described analytical methodology was therefore applied, and the contents and trend variability of myo-inositol in milk from extensively pasture-fed cows during early lactation and across a production season were evaluated. Free and total myo-inositol in seasonal milk were within the ranges of 2.3–4.5 mg 100 g‒1 and 5.3–8.7 mg 100 g‒1, respectively. This new information will both improve understanding of the expression of innate myo-inositol in bovine milk, and provide manufacturers with information that can enhance formulation capability related to the production of cows’ milk-based products.
A method for determining the content of inositol by GC is established, which can be used for the quality control in the process of producing inositol. The relative error of accuracy is 0. 21°, the precision of relative windage is 0.24%, and the recovery rate is 99.93%.
All eight inositols were resolved by liquid chromatography under pressure using a Ca2+-form cation-exchange resin as stationary phase and water as an eluting solvent. The method was successfully applied to the separation of the products formed when myo-inositol was boiled in water in the presence of Raney nickel. In this case, six peaks corresponding to inositols and at least seven unknown peaks were resolved, showing that this liquid chromatographic method is an efficient way to monitor the side reactions which occur during the 1H2H exchange reaction of myo-inositol in deuterium oxide with Raney nickel catalyst.
Each of the inositol diastereomers have been subjected to low resolution gas chromatography‐mass spectrometry as their hexa‐O‐trimethylsilyl (TMSi) ethers and as their hexaacetyl esters. The TMSi ethers of the inositols each possess a unique spectrum consisting of the same ions occurring with remarkable variation in intensity. This variation, which is much greater with the inositol TMSi ethers than in the spectra of the inositol acetates, is presumably an expression of the stereochemistry of each isomer exaggerated by the bulky TMSi groups. Using TMSi‐ d 9 , labeling as well as ring labeling with deuterium we identified several fragment ions which are characteristic of the cyclitols. The spectra of the TMSi inositols are compared with the spectra of other TMSi carbohydrates.
Two main series of fragmentation processes are observed in the inositol hexaacetates. With the aid of acetyl‐ d 3 labeling each of these series were found to divide into two more pathways consisting of ions of the same m / e values but of different structures. These pathways are compared with similar pathways observed in other acetyl carbohydrates.
A method is described for the direct conversion of TMSi ethers to acetate esters which has potential usefulness in natural product studies.
Ten previously unreported oligosaccharides have been purified from the urines of human subjects using a combination of gel filtration, ion exchange, and thin-layer chromatographies. Their structures were determined by direct probe mass spectrometry, methylation analysis, and proton NMR spectroscopy of the permethylated oligosaccharide alditols.
On the basis of composition, the oligosaccharides could be divided into three groups. Five oligosaccharides containing glycerol were characterized as glucosylα1-1′glycerol; glucosylβ1-1′glycerol; galactosylβ1-1′glycerol; glucosyl-1-1′(fucosyl-1-2′)glycerol and/or fucosyl-1-1′(glucosyl-1-2′)glycerol; and glucosyl-1-1′(galactosyl-1-2′)glycerol or galactosyl-1-1′(glucosyl-1-2′)glycerol. Four inositol-containing oligosaccharides were characterized as galactosylβ1 (fucosylα1)inositol,N-acetylgalactosaminylα1 (fucosylα1)inositol, fucosylα1-2galactosylβ1 (N-acetylgalactosaminylα1)inositol and fucosylα1-2galactosylβ1-4-N-acetylglucosaminylα1(N-acetylgalactosaminylα1)inositol. Finally, galactosylα1-3(fucosylα1-2)galactosylβ1-6galactosylα1-4(fucosylα1-3)glucose, an oligosaccharide with glucose at its reducing end, was tentatively identified. The significance and possible origins of the carbohydrate structures are discussed.
A method for the quantitative determination of free and lipid phosphatide myoinositol from brain, liver, and kidney of the rat by gas-liquid chromatography is described. After isolation of the free or lipid-bound cyclohexitol from tissues by conventional techniques, the myoinositol is converted to the hexa-O-trimethylsilyl ether derivative. The method is rapid and sensitive and is not hampered by contaminating carbohydrates. The myoinositol concentrations of the tissues analyzed are in good agreement with those determined by microbiological assay and spectrophotometric procedures. Average recovery of added myoinositol to brain extracts is 98.2%. The gas-chromatographic behavior of 7 inositol isomers and myoinosose-2 on polar and nonpolar columns is reported.
The scrapie (PrPSc) and cellular (PrPC) prion proteins are encoded by the same gene, and their different properties are thought to arise from posttranslational modifications. We have found a phosphatidylinositol glycolipid on both PrPC and PrP 27-30 (derived from PrPSc by limited proteolysis at the amino terminus). Ethanolamine, myo-inositol, phosphate, and stearic acid were identified as glycolipid components of gel-purified PrP 27-30. PrP 27-30 contains 2.8 moles of ethanolamine per mole. Incubation of PrP 27-30 with a bacterial phosphatidylinositol-specific phospholipase C (PIPLC) releases covalently bound stearic acid, and allows PrP 27-30 to react with antiserum specific for the PIPLC-digested glycolipid linked to the carboxyl terminus of the trypanosomal variant surface glycoprotein. PIPLC catalyzes the release of PrPC from cultured mammalian cells into the medium. These observations indicate that PrPC is anchored to the cell surface by the glycolipid.
Several oligosaccharides from human milk were separated completely on a Dionex AS6 ion-exchange column under mild alkaline conditions, such that no degradation of oligosaccharides was detectable after incubation for 6 h at room temperature in the eluent buffer. In general, both the presence of fucosyl groups and branching within oligosaccharide chains tend to reduce retention times for oligosaccharides in this system. Thus, both lacto-N-fucopentaose II [beta-D-Galp-(1----3)-[alpha-L-Fucp-(1----4)]-beta-D-GlcpNAc-(1--- -3)- beta-D-Galp-(1----4)-D-Glc] and lacto-N-fucopentaose III [beta-D-Galp-(1----4)-[alpha-L-Fucp-(1----3)]-beta-D-GlcpNAc-(1--- -3)- beta-D-Galp-(1----4)-D-Glc] were eluted before lacto-N-fucopentaose I [alpha-L-Fucp-(1----2)-beta-D-Galp-(1----3)-beta-D-GlcpNAc-(1----3 )-beta-D- Galp-(1----4)-D-Glc], and all three fucopentaoses were eluted earlier than lacto-N-tetraose [beta-D-Galp-(1----3)-beta-D-GlcpNAc-(1----3)-beta-D-Galp-(1----4)-D-Glc ]. 2'-Fucosyllactose [alpha-L-Fucp-(1----2)-beta-D-Galp-(1----4)-D-Glc] and 3-fucosyllactose [alpha-D-Galp-(1----4)-[alpha-L-Fucp-(1----3)]-D-Glc] were separated completely under a wide range of conditions. Introduction of substituents at either the O-3 or O-4 of 2-acetamido-2-deoxy-D-glucose produced large differences in retention: lacto-N-tetraose and lacto-N-neotetraose [beta-D-Gal-(1----4)-beta-D-GlcpNAc-(1----3)-beta-D-Galp-(1----4)-D-Glc] could be separated by more than 8 min. The l.c. ion-exchange chromatographic system described represents a useful addition to existing methods for separating oligosaccharides derived from glycoproteins and glycolipids.
It has been established that insulin treatment of cells, isolated plasma membranes, or whole animals leads to the generation of low molecular weight mediators which serve as intermediates in the signalling pathway. At least two distinct classes of mediator have been described, based on differences in apparent molecular weight, isoelectric point and biological activity (Cheng, K., and Larner, J. (1985) Ann. Rev. Physiol. 45, 407-424). Recently, Saltiel's (Saltiel, A.R., and Cuatrecasas, P. (1986) Proc. Natl. Acad. Sci. U.S.A. 83, 5793-5797) and Mato's (Mato, J.M., Kelly, K.L., Abler, A., and Jarett, L. (1987) J. Biol. Chem. 262, 2131-2137) laboratories have described an insulin "modulator" which was apparently derived from glycosylphosphoinositol linker, similar to those known to anchor proteins to the external surface of the cell membrane (Low, M.G. (1987) Bioch. J. 244, 1-13). In this paper, we report that highly purified preparations of the insulin mediator which stimulates pyruvate dehydrogenase phosphatase contain mannose, galactosamine, and D-chiroinositol. These determinations are based upon analyses using paper chromatography and gas chromatography/mass spectroscopy. Nitrous acid deamination of the mediator resulted in release of inositol phosphate, indicating that the galactosamine and D-chiroinositol are linked. Although the presence of chiroinositol in modulator from H35 hepatoma cells has been recently reported (Mato, J.M., Kelly, K.L., Abler, A., Jarett, L., Corkey, B.E., Cashel, J.A., and Zopf, D. (1987) Bioch. Biophys. Res. Comm. 146, 764-770), the optical identity of the inositol remained unknown until the present report. Likewise, the presence of galactosamine rather than glucosamine in insulin mediator is a novel finding. These findings, coupled with those of Saltiel and Mato's groups, provide clear evidence for the existence of multiple forms of insulin mediators. Additionally, the results presented here afford further confirmation for the formation of insulin mediators from glycosyl-phosphoinositol linkers.
A class of membrane molecules has been identified whose primary translation product includes a COOH-terminal protein sequence that signals attachment of a glycosyl-phosphatidylinositol anchor at a COOH-terminal residue that is newly formed by cleavage of the signaling sequence. This class includes a wide diversity of protein types from eukaryotes at many stages of evolution. The structures of the glycosyl-phosphatidylinositol anchors are being resolved, but their functions aside from membrane attachment and dynamics remain to be determined.
Many external signals (hormones, neurotransmitters, and growth factors) act through receptors which stimulate the hydrolysis of a minor inositol lipid to give diacylglycerol and inositol trisphosphate. The latter functions by mobilizing calcium from intracellular stores, whereas diacylglycerol stimulates protein kinase C to phosphorylate specific proteins, some of which regulate ionic mechanisms such as the Na⁺/H⁺ exchanger and potassium channels. These two second messenger pathways constitute a highly versatile signaling system controlling numerous cellular processes such as secretion, contraction, metabolism, neuronal excitability, and cell growth.