Phosphatidylinositol 4,5-bisphosphate (PIP2) stimulates the electrogenic Na/HCO3 cotransporter NBCe1-A expressed in Xenopus oocytes

Bicarbonate transporters are regulated by signaling molecules/ions such as protein kinases, ATP, and Ca²⁺. While phospholipids such as PIP₂ can stimulate Na-H exchanger activity, little is known about phospholipid regulation of bicarbonate transporters. We used the patch-clamp technique to study the function and regulation of heterologously expressed rat NBCe1-A in excised macropatches from Xenopus laevis oocytes. Exposing the cytosolic side of inside-out macropatches to a 5% CO₂/33 mM HCO₃⁻ solution elicited a mean inward current of 14 pA in 74% of macropatches attached to pipettes (−V_p = −60 mV) containing a low-Na⁺, nominally HCO₃⁻-free solution. The current was 80–90% smaller in the absence of Na⁺, approximately 75% smaller in the presence of 200 μM DIDS, and absent in macropatches from H₂O-injected oocytes. NBCe1-A currents exhibited time-dependent rundown that was inhibited by removing Mg²⁺ in the presence or absence of vanadate and F⁻ to reduce general phosphatase activity. Applying 5 or 10 μM PIP₂ (diC8) in the presence of HCO₃⁻ induced an inward current in 54% of macropatches from NBC-expressing, but not H₂O-injected oocytes. PIP₂-induced currents were HCO₃⁻-dependent and somewhat larger following more NBCe1-A rundown, 62% smaller in the absence of Na⁺, and 90% smaller in the presence of 200 μM DIDS. The polycation neomycin (250–500 μM) reduced the PIP₂-induced inward current by 69%; spermine (100 μM) reduced the current by 97%. Spermine, poly-D-lysine, and neomycin all reduced the baseline HCO₃⁻-induced inward currents by as much as 85%. In summary, PIP₂ stimulates NBCe1-A activity, and phosphoinositides are regulators of bicarbonate transporters.     

Characterization of oxytocin receptors and serotonin transporters in mast cells

Oxytocin (OT) inhibits the uptake of serotonin (5HT) into uterine mast cells. This may modulate 5HT bioavailability in the myometrium. Because 5HT is an important endogenous uterotonic compound, it has been postulated that this effect of OT may contribute to its potency as a labor inducer. This also predicts the presence of oxytocin receptors (OTRs) and transducing signals that will interact with 5HT transporters (SERT) in mast cells. In this study, OTR and SERT were characterized in murine peritoneal mast cells by radioligandbinding studies. Saturation assays for OTR showed no changes in K _d along the estrous cycle (6.95±2.76 nM in estrus and 4.07±1.73 nM in diestrus) but an increase in B _max in estrus (0.71±0.08 pmol/10⁶ cells and 0.37±0.05 pmol/10⁶ cells in estrus and diestrus, respectively). B _max and K _d for SERT were not affected along the estrous cycle. The signaling between the OTR and the SERT was analyzed by measuring the extent of inhibition of OT and PMA (activator of protein kinase C on 5HT uptake and the capability of Ro318220 (specific inhibitor of PKC) to increase 5HT uptake and block the effect of the above compounds in mast cells. The results showed that in murine peritoneal mast cells in vitro (1) ovarian hormones modulate OTR but not SERT expression, (2) the magnitude of OT action on 5HT uptake depends on the number of OTRs expressed in mast cells, and (3) the signaling between OTR and the SERT is mediated through the activation of protein kinase C. It is concluded that the ovarian hormones have a modulatory action on 5HT uptake which involves OT-mediated mechanism.     

Alterations of phosphoinositide-specific phospholipase C and protein kinase C in the myocardium of spontaneously hypertensive rats

Summary In order to determine whether phosphoinositide metabolism is altered in hypertensive cardiac hypertrophy, phospholipase C (PLC) and protein kinase C activities were measured in hearts from 4- and 20-week-old spontaneously hypertensive rats (SHR) and age-matched, normotensive Wister-Kyoto rats (WKY). PLC activities were assayed using phosphatidylinositol (PI) and phosphatidylinositol-4,5-bisphosphate (PIP₂) as substrates to assess the substrate specificity. PI-hydrolyzing PLC activity (PI-PLC) was predominantly located in the cytosol, and its activity was similar in both strains. Membrane-bound PIP₂-hydrolyzing PLC activity (PIP₂-PLC) was significantly lower in 20-week-old SHR than in WKY, but there was no significant difference in soluble PIP₂-PLC. Protein kinase C activity was significantly elevated in 20-week-old SHR and Ca²⁺-phospholipid-dependent phosphorylation was observed in the proteins of molecular weight 26, 32, 43, and 95 KDa. In 4-week-old prehypertensive SHR, there were no significant differences in PI-PLC, PIP₂-PLC, or protein kinase C activities as compared with age-matched WKY. These data demonstrated that protein kinase C and membrane-bound PIP₂-PLC are altered during the period of hypertension development. These alterations may have important roles in the development or maintenance of hypertensive cardiac hypertrophy in SHR.Until April 31, 1990 N. Makita, Department of Cardiovascular Medicine, Hokkaido University School of Medicine, Kita 15, Nishi 7, Kita-Ku Sapporo 060, Japan After May 1, 1990 N. Makita, Division of Nephrology, S-3223, Medical Center North, Vanderbilt University, Nashville. Tennessee 37232, USA     

Melatonin inhibits serotonin transporter activity in intestinal epithelial cells

Abstract: Gastrointestinal serotonin (5‐HT) and melatonin are two closely related neuromodulators which are synthesised in the enterochromaffin cells of the intestinal epithelium and which have been shown to be involved in the physiopathology of the gastrointestinal tract. The effects of 5‐HT depend on 5‐HT availability which is, in part, modulated by the serotonin transporter (SERT). This transporter provides an efficient 5‐HT uptake after release and is expressed in the membrane of the enterocytes. Although the origin and effects of 5‐HT and melatonin are similar, the interrelationship between them in the gastrointestinal tract is unknown. The main aim of this study was to determine whether melatonin affects SERT activity and expression, and, if so, to elucidate the mechanisms involved. Caco‐2 cell line was used to carry out the study as these cells have been shown to endogenously express SERT. The results showed that melatonin inhibits SERT activity by affecting both V_max and kt kinetic constants although SERT synthesis or intracellular trafficking did not appear to be affected. The melatonin effect seemed to be independent of melatonin receptors MT₁ and MT₂ and protein kinase C and cAMP intracellular pathways. Our results suggest that the inhibition of SERT might be due to a catalytic effect of melatonin on the allosteric citalopram‐sensitive site in SERT. This study shows, for the first time, that melatonin modulates SERT activity, thus demonstrating the feedback system between melatonin and the serotoninergic system in the gastrointestinal tract.     

A single serine residue controls the cation dependence of substrate transport by the rat serotonin transporter

The serotonin transporter (SERT) is a member of the Na⁺/Cl⁻-dependent neurotransmitter transporter family and constitutes the target of several clinically important antidepressants. Here, replacement of serine-545 in the recombinant rat SERT by alanine was found to alter the cation dependence of serotonin uptake. Substrate transport was now driven as efficiently by LiCl as by NaCl without significant changes in serotonin affinity. Binding of the antidepressant [³H]imipramine occurred with 1/5th the affinity, whereas [³H]citalopram binding was unchanged. These results indicate that serine-545 is a crucial determinant of both the cation dependence of serotonin transport by SERT and the imipramine binding properties of SERT.     

Molecular mechanisms of cocaine reward: combined dopamine and serotonin transporter knockouts eliminate cocaine place preference

Cocaine blocks uptake by neuronal plasma membrane transporters for dopamine (DAT), serotonin (SERT), and norepinephrine (NET). Cocaine reward/reinforcement has been linked to actions at DAT or to blockade of SERT. However, knockouts of neither DAT, SERT, or NET reduce cocaine reward/reinforcement, leaving substantial uncertainty about cocaine's molecular mechanisms for reward. Conceivably, the molecular bases of cocaine reward might display sufficient redundancy that either DAT or SERT might be able to mediate cocaine reward in the other's absence. To test this hypothesis, we examined double knockout mice with deletions of one or both copies of both the DAT and SERT genes. These mice display viability, weight gain, histologic features, neurochemical parameters, and baseline behavioral features that allow tests of cocaine influences. Mice with even a single wild-type DAT gene copy and no SERT copies retain cocaine reward/reinforcement, as measured by conditioned place-preference testing. However, mice with no DAT and either no or one SERT gene copy display no preference for places where they have previously received cocaine. The serotonin dependence of cocaine reward in DAT knockout mice is thus confirmed by the elimination of cocaine place preference in DAT/SERT double knockout mice. These results provide insights into the brain molecular targets necessary for cocaine reward in knockout mice that develop in their absence and suggest novel strategies for anticocaine medication development.     

Physical interaction between the serotonin transporter and neuronal nitric oxide synthase underlies reciprocal modulation of their activity

The spatiotemporal regulation of neurotransmitter transporters involves proteins that interact with their intracellular domains. Using a proteomic approach, we identified several proteins that interact with the C terminus of the serotonin transporter (SERT). These included neuronal nitric oxide synthase (nNOS), a PSD-95/Disc large/ZO-1 (PDZ) domain-containing protein recruited by the atypical PDZ binding motif of SERT. Coexpression of nNOS with SERT in HEK293 cells decreased SERT cell surface localization and 5-hydroxytryptamine (5-HT) uptake. These effects were absent in cells transfected with SERT mutated in its PDZ motif to prevent physical association with nNOS, and 5-HT uptake was unaffected by activation or inhibition of nNOS enzymatic activity. 5-HT uptake into brain synaptosomes was increased in both nNOS-deficient and wild-type mice i.v. injected with a membrane-permeant peptidyl mimetic of SERT C terminus, which disrupted interaction between SERT and nNOS, suggesting that nNOS reduces SERT activity in vivo. Furthermore, treating cultured mesencephalic neurons with the mimetic peptide similarly increased 5-HT uptake. Reciprocally, indicating that 5-HT uptake stimulates nNOS activity, NO production was enhanced on exposure of cells cotransfected with nNOS and SERT to 5-HT. This effect was abolished by 5-HT uptake inhibitors and absent in cells expressing SERT mutated in its PDZ motif. In conclusion, physical association between nNOS and SERT provides a molecular substrate for their reciprocal functional modulation. In addition to showing that nNOS controls cell surface localization of SERT, these findings provide evidence for regulation of cellular signaling (NO production) by a substrate-carrying transporter.     

Phosphatidylinositol 4,5-bisphosphate alters pharmacological selectivity for epilepsy-causing KCNQ potassium channels

Pharmacological augmentation of neuronal KCNQ muscarinic (M) currents by drugs such as retigabine (RTG) represents a first-in-class therapeutic to treat certain hyperexcitatory diseases by dampening neuronal firing. Whereas all five potassium channel subtypes (KCNQ1–KCNQ5) are found in the nervous system, KCNQ2 and KCNQ3 are the primary players that mediate M currents. We investigated the plasticity of subtype selectivity by two M current effective drugs, retigabine and zinc pyrithione (ZnPy). Retigabine is more effective on KCNQ3 than KCNQ2, whereas ZnPy is more effective on KCNQ2 with no detectable effect on KCNQ3. In neurons, activation of muscarinic receptor signaling desensitizes effects by retigabine but not ZnPy. Importantly, reduction of phosphatidylinositol 4,5-bisphosphate (PIP₂) causes KCNQ3 to become sensitive to ZnPy but lose sensitivity to retigabine. The dynamic shift of pharmacological selectivity caused by PIP₂ may be induced orthogonally by voltage-sensitive phosphatase, or conversely, abolished by mutating a PIP₂ site within the S4–S5 linker of KCNQ3. Therefore, whereas drug-channel binding is a prerequisite, the drug selectivity on M current is dynamic and may be regulated by receptor signaling pathways via PIP₂.     

Characterization of cytoplasmic and membrane-associated phosphatidylinositol 4,5-biphosphate phospholipase C activities in guinea pig ventricles

Summary The phosphoinositide-specific phospholipase C (PLC) activity present in the soluble and sarcolemmal enriched membrane fraction from guinea pig hearts was characterized using phosphatidyl [³H]inositol 4,5-biphosphate (PIP₂) or phosphatidyl [³H]inositol 4-monophosphate (PIP) as substrates. The PLC activities (cytosolic and membrane associated) were specific for polyphosphoinositides (PIP₂ and PIP) since no other phospholipids were hydrolyzed at pH 7.0 under various ionic conditions. Both enzymic activities were Ca²⁺-dependent (half maximal activities were achieved around pCa 5.0). The pH, detergent (deoxycholate), divalent (Ca²⁺ and Mg²⁺), and monovalent (Na⁺ and K⁺) cation dependencies were very similar between the cytosolic and membrane-associated enzyme activities, using either PIP₂ or PIP as substrate. Hydrolysis of the polyphosphoinositides was inhibited in the presence of phosphatidylethanolamine, phosphatidylserine, or phosphatidylcholine.Under optimal conditions (pH 7.0, 1 mM Ca²⁺, 2.5 mM Mg²⁺, 100 mM Na⁺ and 0.07% deoxycholate) the specific activities of the cytosolic and membrane-associated enzymes were 19.9±0.9 and 10.1±0.9 nmol/min/mg protein, respectively, using PIP₂ as substrate. Under the same conditions these activitics were 18.1±1.0 and 8.0±0.8 nmol/min/mg protein for the cytosolic and membrane fractions, respectively, using PIP as substrate. Based on the similarity of the characteristics of these two PLC enzyme activities, it is suggested that the cytosolic and membrane-associated enzyme forms may be closely related.This work was supported by National Institutes of Health Grants HL26057 and HL22619.     

Kv7.1 ion channels require a lipid to couple voltage sensing to pore opening

Voltage-gated ion channels generate dynamic ionic currents that are vital to the physiological functions of many tissues. These proteins contain separate voltage-sensing domains, which detect changes in transmembrane voltage, and pore domains, which conduct ions. Coupling of voltage sensing and pore opening is critical to the channel function and has been modeled as a protein–protein interaction between the two domains. Here, we show that coupling in Kv7.1 channels requires the lipid phosphatidylinositol 4,5-bisphosphate (PIP₂). We found that voltage-sensing domain activation failed to open the pore in the absence of PIP₂. This result is due to loss of coupling because PIP₂ was also required for pore opening to affect voltage-sensing domain activation. We identified a critical site for PIP₂-dependent coupling at the interface between the voltage-sensing domain and the pore domain. This site is actually a conserved lipid-binding site among different K⁺ channels, suggesting that lipids play an important role in coupling in many ion channels.     

A novel form of immune signaling revealed by transmission of the inflammatory mediator serotonin between dendritic cells and T cells

Adaptive immunity is triggered at the immune synapse, where peptide-major histocompatibility complexes and costimulatory molecules expressed by dendritic cells (DCs) are physically presented to T cells. Here we describe transmission of the inflammatory monoamine serotonin (5-hydroxytryptamine [5-HT]) between these cells. DCs take up 5-HT from the microenvironment and from activated T cells (that synthesize 5-HT) and this uptake is inhibited by the antidepressant, fluoxetine. Expression of 5-HT transporters (SERTs) is regulated by DC maturation, exposure to microbial stimuli, and physical interactions with T cells. Significantly, 5-HT sequestered by DCs is stored within LAMP-1+ vesicles and subsequently released via Ca2+-dependent exocytosis, which was confirmed by amperometric recordings. In turn, extracellular 5-HT can reduce T-cell levels of cAMP, a modulator of T-cell activation. Thus, through the uptake of 5-HT at sites of inflammation, and from activated T cells, DCs may shuttle 5-HT to naive T cells and thereby modulate T-cell proliferation and differentiation. These data constitute the first direct measurement of triggered exocytosis by DCs and reveal a new and rapid type of signaling that may be optimized by the intimate synaptic environment between DCs and T cells. Moreover, these results highlight an important role for 5-HT signaling in immune function and the potential consequences of commonly used drugs that target 5-HT uptake and release.     

Ceramide kinase regulates phospholipase C and phosphatidylinositol 4, 5, bisphosphate in phototransduction

Phosphoinositide-specific phospholipase C (PLC) is a central effector for many biological responses regulated by G-protein–coupled receptors including Drosophila phototransduction where light sensitive channels are activated downstream of NORPA, a PLCβ homolog. Here we show that the sphingolipid biosynthetic enzyme, ceramide kinase, is a novel regulator of PLC signaling and photoreceptor homeostasis. A mutation in ceramide kinase specifically leads to proteolysis of NORPA, consequent loss of PLC activity, and failure in light signal transduction. The mutant photoreceptors also undergo activity-dependent degeneration. Furthermore, we show that a significant increase in ceramide, resulting from lack of ceramide kinase, perturbs the membrane microenvironment of phosphatidylinositol 4, 5, bisphosphate (PIP₂), altering its distribution. Fluorescence image correlation spectroscopic studies on model membranes suggest that an increase in ceramide decreases clustering of PIP₂ and its partitioning into ordered membrane domains. Thus ceramide kinase–mediated maintenance of ceramide level is important for the local regulation of PIP₂ and PLC during phototransduction.Signal transduction via G-protein–coupled receptors (GPCRs) is vital for many cellular processes including vision, olfaction, taste, and neurotransmission. Extensive studies on proteins constituting this family and their interactions reveal complex signaling networks regulated at multiple levels (1). Lipids play equally important roles in GPCR signaling, as most of the signal transduction machinery is membrane associated. How lipids regulate GPCR signaling is being addressed in recent years (2). Our current knowledge of how lipids influence each other to form membrane microenvironments and how this modulates proteins during signal transduction in a multicellular organism is limited. In this study, we address this issue in the context of Drosophila phototransduction, a prototypic G-protein–coupled phosphoinositide cascade, by genetically modulating the sphingolipid ceramide.Analyses of Drosophila phototransduction have led to the identification, characterization, and regulation of many signaling components (3). Phototransduction begins with the absorption of light by rhodopsin, followed by the activation of a G protein (Gα_q). Gα_q activates the critical effector NORPA, a phospholipase C (PLC) that catalyzes the hydrolysis of phosphatidylinositol 4, 5-bisphosphate (PIP₂) into two important second messengers, diacylglycerol and inositol 1, 4, 5-trisphosphate. Activation of PLC leads to gating of two transduction channels, transient receptor potential (TRP) and TRP-like. Although many of the proteins involved in phototransduction have been well characterized, we are only beginning to understand how lipids and enzymes involved in lipid metabolism regulate this cascade (4–7). Sphingolipids are integral components of all eukaryotic cell membranes and also act as second messengers in diverse signaling pathways (8). The sphingolipid biosynthetic pathway is an evolutionarily conserved route that generates and interconverts various sphingolipids such as ceramide, sphingosine, ceramide 1–phosphate and sphingosine 1–phosphate (9). We showed earlier that modulating this biosynthetic pathway by targeted overexpression of Drosophila neutral ceramidase (CDase), an enzyme that converts ceramide to sphingosine, rescues retinal degeneration in an arrestin mutant, and facilitates membrane turnover in a rhodopsin null mutant by modulating the endocytic machinery (10–12). Although these studies established that ceramide metabolism is important for survival of photoreceptors, they did not evaluate its role in signaling events during phototransduction.Ceramide kinase (CERK), a recently cloned lipid kinase, phosphorylates ceramide to ceramide 1–phosphate (C-1-P), thereby decreasing ceramide levels (13, 14). Here we show that Drosophila ceramide kinase (DCERK) regulates PLC activity, function, and the local organization of PIP₂ in GPCR signaling by controlling the ceramide level. Genetic, biochemical, and electrophysiological analyses of DCERK deficient flies reveal a severe down regulation of NORPA and failure in phototransduction. Increased ceramide levels in the mutant also alter the level and membrane microenvironment of PIP₂ that correlates with a failure of NORPA to localize to the membranes. Using fluorescence image correlation spectroscopy in supported bilayers, we show that ceramide perturbs both the protein-dependent and -independent compartmentalization of PIP₂, thus providing a biophysical basis for the effect of ceramide on PIP₂. These findings show that sphingolipids and phospholipids cooperate in vivo to establish a suitable membrane microenvironment for signaling mediated by PLC.     

A biobehavioural model of the night eating syndrome

This paper will propose a biobehavioral mechanism for the Night Eating Syndrome (NES), a disorder characterized by a delayed circadian rhythm of food intake and neuroendocrine function. Food intake consists of at least 25% of daily caloric intake after the evening meal and/or at least two nighttime awakenings with ingestions per week. This will be explored by reviewing neuroimaging of brain serotonin transporters (SERT) and treatment with selective serotonin reuptake inhibitors (SSRIs). SERT binding is elevated in the midbrain of night eaters, causing dysregulation of the circadian rhythm of both food intake and neuroendocrine function. The administration of SSRIs blocks the reuptake of serotonin and restores the circadian rhythm of both food intake and neuroendocrine function. This hypothesis implies that reduction of SERT activity should increase postsynaptic serotonin transmission and relieve NES. This is precisely the effect of SSRIs. NES is a function of elevated SERT, and blocking of SERT with an SSRI resolves NES. This model of NES attests to the validity of the diagnosis of NES and the criteria by which it is identified, and it provides an explanation of the mechanism.     

Phosphatidylinositol-4,5-biphosphate-dependent rearrangement of TRPV4 cytosolic tails enables channel activation by physiological stimuli

Most transient receptor potential (TRP) channels are regulated by phosphatidylinositol-4,5-biphosphate (PIP₂), although the structural rearrangements occurring on PIP₂ binding are currently far from clear. Here we report that activation of the TRP vanilloid 4 (TRPV4) channel by hypotonic and heat stimuli requires PIP₂ binding to and rearrangement of the cytosolic tails. Neutralization of the positive charges within the sequence ¹²¹KRWRK¹²⁵, which resembles a phosphoinositide-binding site, rendered the channel unresponsive to hypotonicity and heat but responsive to 4α-phorbol 12,13-didecanoate, an agonist that binds directly to transmembrane domains. Similar channel response was obtained by depletion of PIP₂ from the plasma membrane with translocatable phosphatases in heterologous expression systems or by activation of phospholipase C in native ciliated epithelial cells. PIP₂ facilitated TRPV4 activation by the osmotransducing cytosolic messenger 5′-6’-epoxyeicosatrienoic acid and allowed channel activation by heat in inside-out patches. Protease protection assays demonstrated a PIP₂-binding site within the N-tail. The proximity of TRPV4 tails, analyzed by fluorescence resonance energy transfer, increased by depleting PIP₂ mutations in the phosphoinositide site or by coexpression with protein kinase C and casein kinase substrate in neurons 3 (PACSIN3), a regulatory molecule that binds TRPV4 N-tails and abrogates activation by cell swelling and heat. PACSIN3 lacking the Bin-Amphiphysin-Rvs (F-BAR) domain interacted with TRPV4 without affecting channel activation or tail rearrangement. Thus, mutations weakening the TRPV4–PIP₂ interacting site and conditions that deplete PIP₂ or restrict access of TRPV4 to PIP₂—in the case of PACSIN3—change tail conformation and negatively affect channel activation by hypotonicity and heat.     

Epidermal growth factor upregulates serotonin transporter and its association with visceral hypersensitivity in irritable bowel syndrome

AIM: To investigate the role of epidermal growth factor (EGF) in visceral hypersensitivity and its effect on the serotonin transporter (SERT).METHODS: A rat model for visceral hypersensitivity was established by intra-colonic infusion of 0.5% acetic acid in 10-d-old Sprague-Dawley rats. The visceral sensitivity was assessed by observing the abdominal withdrawal reflex and recording electromyographic activity of the external oblique muscle in response to colorectal distension. An enzyme-linked immunosorbent assay was used to measure the EGF levels in plasma and colonic tissues. SERT mRNA expression was detected by real-time PCR while protein level was determined by Western blot. The correlation between EGF and SERT levels in colon tissues was analyzed by Pearson’s correlation analysis. SERT function was examined by tritiated serotonin (5-HT) uptake experiments. Rat intestinal epithelial cells (IEC-6) were used to examine the EGF regulatory effect on SERT expression and function via the EGF receptor (EGFR).RESULTS: EGF levels were significantly lower in the rats with visceral hypersensitivity as measured in plasma (2.639 ± 0.107 ng/mL vs 4.066 ± 0.573 ng/mL, P < 0.01) and in colonic tissue (3.244 ± 0.135 ng/100 mg vs 3.582 ± 0.197 ng/100 mg colon tissue, P < 0.01) compared with controls. Moreover, the EGF levels were positively correlated with SERT levels (r = 0.820, P < 0.01). EGF displayed dose- and time-dependent increased SERT gene expressions in IEC-6 cells. An EGFR kinase inhibitor inhibited the effect of EGF on SERT gene upregulation. SERT activity was enhanced following treatment with EGF (592.908 ± 31.515 fmol/min per milligram vs 316.789 ± 85.652 fmol/min per milligram protein, P < 0.05) and blocked by the EGFR kinase inhibitor in IEC-6 cells (590.274 ± 25.954 fmol/min per milligram vs 367.834 ± 120.307 fmol/min per milligram protein, P < 0.05).CONCLUSION: A decrease in EGF levels may contribute to the formation of visceral hypersensitivity through downregulation of SERT-mediated 5-HT uptake into enterocytes.     

Polyphosphoinositide Changes in Rabbit Platelets Stimulated with Platelet Activating Factor During the Formation of Platelet-fibrin Clots

Phosphoinositide metabolism in rabbit platelets prelabelled with [³²P]phosphate and [³H]inositol was stimulated by platelet activating factor (PAF, 1-0-alkyl-2-acetyl-sn-glyceryl-3-phosphorylcholine) with stirring at 200 rpm for 120 s in the presence of polymerising fibrin produced by the action of batroxobin (B. atrox) (also referred to by the proprietary name Reptilase) on fibrinogen. Under these conditions platelet-fibrin clots formed and retracted around the stirring bar. Phosphoinositides were extracted with chloroform: methanol: HC1. The role of the secretion of platelet granule contents in the phosphoinositide changes was examined by comparison of the effects of 1 nM PAF which did not cause secretion, with 50 nM PAF which caused extensive secretion. Stimulation of platelets with PAF in the presence of polymerising fibrin caused a greater decrease in the amount and labelling of extractable phosphatidylinositol 4,5-bisphosphate (PIP₂) than was observed with platelets stimulated in the presence of fibrinogen. With 1 nM PAF, the decrease (1.26 ± 0.11 nmol/10⁹ platelets) in amount of extractable PIP₂ when platelets were stimulated in the presence of polymerising fibrin compared with in the presence of fibrinogen was accounted for by an increase in the amount of phosphatidylinositol 4-phosphate (PIP). With 50 nM PAF, the decrease in amount of extractable PIP₂ (1.09±0.11 nmol/10⁹ platelets) was not accounted for by an increase in the amount of PIP; the decrease in the amount of [³H]inositol label in PIP₂ in platelets stimulated in the presence of polymerising fibrin was accounted for by the sum of the increases in PIP labelling and the label associated with interfacial protein from the lipid extractions. When fibrin polymerisation was blocked with glycyl-L-prolyl-L-arginyl-L-proline (GPRP), the large decrease in extractable PIP₂ and the increase in the association of label with the interfacial protein did not occur. Thus, both the formation of a fibrin network, and the changes that accompany the secretion of granule contents, are necessary for the association of the ³H-labelled material with interfacial protein. Blocking thromboxane A₂ formation had no effect on the changes in response to 50 nM PAF. Although PAF stimulated phospholipase C, resulting in increases in amount and ³²P-labelling of phosphatidic acid and ³H-labelling of inositol bisphosphate and inositol phosphate, the increases were similar in the presence of polymerising fibrin or fibrinogen. Thus, further stimulation of phospholipase C does not occur in association with clot formation. The specific radioactivities of labelling with [³H]inositol of the phosphoinositides in unstimulated platelets differed (PIP₂> phosphatidylinositol (PI) > PIP). Upon stimulation of the platelets with 1 nM PAF, the specific radioactivity of PIP rose above that of PI and toward that of PIP₂, indicating that the increase in PIP was due to degradation of PIP₂. Thus, the large decrease in extractable PIP₂ and increase in formation of PIP caused by the presence of polymerising fibrin appear to be due to increased degradation of PIP₂ to PIP.     

Ins(1,4,5)P(3) regulates phospholipase Cbeta1 expression in cardiomyocytes

The functional significance of the Ca²⁺-releasing second messenger inositol(1,4,5)trisphosphate (Ins(1,4,5)P₃, IP₃) in the heart has been controversial. Ins(1,4,5)P₃ is generated from the precursor lipid phosphatidylinositol(4,5)bisphosphate (PIP₂) along with sn-1,2-diacylglycerol, and both of these are important cardiac effectors. Therefore, to evaluate the functional importance of Ins(1,4,5)P₃ in cardiomyocytes (NRVM), we overexpressed IP₃ 5-phosphatase to increase degradation. Overexpression of IP₃ 5-phosphatase reduced Ins(1,4,5)P₃ responses to α₁-adrenergic receptor agonists acutely, but with longer stimulation, caused an overall increase in phospholipase C (PLC) activity, associated with a selective increase in expression of PLCβ1, that served to normalise Ins(1,4,5)P₃ content. Similar increases in PLC activity and PLCβ1 expression were observed when Ins(1,4,5)P₃ was sequestered onto the PH domain of PLCδ1, a high affinity selective Ins(1,4,5)P₃-binding motif. These findings suggested that the available level of Ins(1,4,5)P₃ selectively regulates the expression of PLCβ1. Cardiac responses to Ins(1,4,5)P₃ are mediated by type 2 IP₃-receptors. Hearts from IP₃-receptor (type 2) knock-out mice showed heightened PLCβ1 expression. We conclude that Ins(1,4,5)P₃ and IP₃-receptor (type 2) regulate PLCβ1 and thereby maintain levels of Ins(1,4,5)P₃. This implies some functional significance for Ins(1,4,5)P₃ in the heart.     

Lubiprostone Targets Prostanoid Signaling and Promotes Ion Transporter Trafficking, Mucus Exocytosis, and Contractility

Background and Aim

Lubiprostone is a chloride channel activator in clinical use for the treatment of chronic constipation, but the mechanisms of action of the drug are poorly understood. The aim of this study was to determine whether lubiprostone exerts secretory effects in the intestine by membrane trafficking of ion transporters and associated machinery.

Methods

Immunolabeling and quantitative fluorescence intensity were used to examine lubiprostone-induced trafficking of the cystic fibrosis transmembrane conductance regulator (CFTR), sodium/potassium-coupled chloride co-transporter 1 (NKCC1), electrogenic sodium/bicarbonate co-transporter 1 (NBCe1), down-regulated in adenoma (DRA), putative anion transporter 1 (PAT1), sodium/proton exchanger 3 (NHE3), Ca²⁺ activated chloride channel 2 (ClC-2) serotonin and its transporter SERT, E prostanoid receptors EP4 and EP1, sodium/potassium ATPase (Na–K-ATPase) and protein kinase A (PKA). The effects of lubiprostone on mucus exocytosis in rat intestine and human rectosigmoid explants were also examined.

Results

Lubiprostone induced contraction of villi and proximal colonic plicae and membrane trafficking of transporters that was more pronounced in villus/surface cells compared to the crypt. Membrane trafficking was determined by: (1) increased membrane labeling for CFTR, PAT1, NKCC1, and NBCe1 and decreased membrane labeling for NHE3, DRA and ClC-2; (2) increased serotonin, SERT, EP4, EP1 and PKA labeling in enterochromaffin cells; (3) increased SERT, EP4, EP1, PKA and Na–K-ATPase in enterocytes; and (4) increased mucus exocytosis in goblet cells.

Conclusion

These data suggest that lubiprostone can target serotonergic, EP4/PKA and EP1 signaling in surface/villus regions; stimulate membrane trafficking of CFTR/NBCe1/NKCC1 in villus epithelia and PAT1/NBCe1/NKCC1 in colonic surface epithelia; suppress NHE3/DRA trafficking and fluid absorption; and enhance mucus-mobilization and mucosal contractility.     

The effect of pH on 86Rubidium efflux from pancreatic islet cells

The influence of pH on the K⁺ permeability of pancreatic islet cells was investigated by measuring ⁸⁶Rb⁺ fluxes in isolated rat islets perifused or incubated in the absence of glucose. Acidification of the medium (to pH 6.5), by decreasing the concentration of HCO^?₃ or by increasing pCO₂ at constant HCO^?₃ reversibly reduced the rate of ⁸⁶Rb⁺ efflux from perifused islets. Alkalinization of the medium (to pH 7.8), by decreasing the pCO₂, reversibly increased ⁸⁶Rb⁺ efflux. Similar changes were recorded upon alteration of the pH in a bicarbonate-free, Hepesbuffered medium with or without calcium. Alteration of the CO₂ level at constant external pH — in order to modify internal pH — produced only a small and transient increase in efflux rate when CO₂ was lowered, and a decrease in efflux rate when CO₂ was raised. NH₄Cl reversibly augmented ⁸⁶Rb⁺ efflux in the presence and in the absence of HCO^?₃. At low pH (6.5), ⁸⁶Rb⁺ uptake by islet cells was reduced after 10 min (25%) and 30 min (11%), but not after 60 min, of incubation; it was not significantly affected by high pH (7.8). Calcium uptake and insulin release were reduced at low pH and increased at high pH.These results show that the K⁺ permeability of islet cells is affected by changes in extracellular but not in intracellular pH. They suggest that the endogenous production of protons that accompanies glucose stimulation of islet cells is not the mediator of the decrease in K⁺ permeability induced by the sugar.