Evidence for inverse agonism of SR141716A at human recombinant cannabinoid CB1 and CB2 receptors

OBJECTIVES: We studied the clinical spectrum, histology, and malignant potential of colonic polyps in Indian children (< or =12 yr). METHODS: Two hundred thirty-six children with colonic polyps were studied from January 1991 to October 1996. They were evaluated clinically and colonoscopic polypectomy was done. Children with five or more juvenile polyps were labeled as having juvenile polyposis and serial colonoscopic polypectomies were done every 3 wk. Colectomy was performed when there were intractable symptoms or clearing of the polyps by colonoscopy was not possible. Histological examination of the polyps was done. Follow-up colonoscopy was done in children with juvenile polyposis only. RESULTS: The mean age of these children was 6.12 +/- 2.7 yr, with a male preponderance (3.5:1). Rectal bleeding of a mean duration of 14 +/- 16 months was the presenting symptom in 98.7%. Solitary polyps were seen in 76%, multiple polyps in 16.5%, and juvenile polyposis in 7% (n = 17) of the children. A majority (93%) of the polyps were juvenile and 85% were rectosigmoid in location. Adenomatous changes, seen in 11%, were more common in juvenile polyposis (59%) than in juvenile polyps (5%). Among those with juvenile polyposis, colon clearance was achieved in eight, six required colectomy for intractable symptoms, and three were still on the polypectomy program. Polyps recurred in 5% of children with juvenile polyps and 37.5% of those with juvenile polyposis. CONCLUSIONS: Juvenile polyps remain the most common colonic polyps in children. A significant number of cases of polyps are multiple and proximally located, which emphasizes the need for total colonoscopy in all. Juvenile polyps should be removed even if asymptomatic because of their neoplastic potential. Colonoscopic polypectomy is effective even in juvenile polyposis. Surveillance colonoscopy is required in juvenile polyposis only.     

SR 144528, an antagonist for the peripheral cannabinoid receptor that behaves as an inverse agonist

In the present report, we investigated in detail the effects of SR 144528, a selective antagonist of the peripheral cannabinoid receptor (CB2), on two well-characterized functions mediated by CB2: the induction of the early response gene krox24 and the inhibition of adenylyl cyclase. We generated Chinese hamster ovary cells doubly transfected with human CB2 and a luciferase reporter gene linked to either the murine krox24 regulatory sequence or multiple cAMP responsive elements. Our results show that (1) SR 144528 antagonizes the effect of receptor agonists-it inhibits the krox24 reporter activity and prevents the inhibition of forskolin-induced cAMP reporter activity mediated by CP 55,940; (2) CB2 is autoactivated-CB2 mediates signaling in the absence of ligand, and this basal activity is reduced by pretreating the cells with pertussis toxin; (3) SR 144528 is an inverse agonist-it reproduces the effects of pertussis toxin; and (4) inhibition of precoupled CB2 by a long-term pretreatment of cells with SR 144528 potentiates krox24 response to cannabinoid receptor agonists and restores activation of adenylyl cyclase. Taken together, these data provide evidences for the inverse agonist property of SR 144528 and the constitutive activation of CB2 in Chinese hamster ovary-expressing cells.     

Antagonism between the anti-inflammatory activity of the cannabinoid WIN 55212-2 and SR 141716A

The CB1/CB2 receptor agonist WIN 55212-2 (0.75 mg/kg, i.v.) caused a significant reduction in neurogenic plasma extravasation induced by electrical stimulation of the saphenous nerve in anesthetized rats; WIN 55212-2 at 2.5-10 mg/kg, s.c., also produced a significant reduction in the carrageenan-induced paw edema in conscious rats. The selective CB1 antagonist SR 141716A (0.075-0.75 mg/kg i.v.) antagonized the WIN 55212-2 effects in the plasma extravasation model and antagonized the WIN 55212-2 (2.5 mg/kg, s.c.)-induced decreases in rectal temperature and increases in tail-flick latencies. However, SR 141716A (10 mg/kg, p.o.) failed to antagonize the effects of Win 55212-2 (2.5 mg/kg, s.c.) in the carrageenan model, suggesting that cannabinoid receptors found in the periphery may be able to modulate inflammatory processes in rats.     

Electrically evoked acetylcholine release from hippocampal slices is inhibited by the cannabinoid receptor agonist, WIN 55212-2, and is potentiated by the cannabinoid antagonist, SR 141716A

This study examined the effect of the cannabinoid receptor agonist, WIN 55212-2, on the electrically evoked release of [14C]acetylcholine (ACh) from superfused brain slices from the hippocampus, a region with a high density of cannabinoid receptors. A comparison was also made with [14C]ACh release from the nucleus accumbens, which has relatively fewer cannabinoid receptors. In the hippocampal slices, WIN 55212-2 produced a dose-dependent inhibition of [14C]ACh release, with an EC50 of 0.03 microM and a maximal inhibition of 81% at 1 microM. In the nucleus accumbens slices, WIN 55212-2 produced a weak inhibition of [14C]ACh release, which did not quite reach statistical significance. The inhibition of electrically evoked hippocampal [14C]ACh release by WIN 55212-2 could be prevented by the cannabinoid receptor antagonist, SR 141716A (EC50, 0.3-1.0 microM). In addition to antagonizing the effects of WIN 55212-2, SR 141716A alone produced a 2-fold potentiation of the electrically stimulated [14C]ACh release in this region (EC50, 0.1-0.3 microM). By contrast, in nucleus accumbens slices, no potentiation of the stimulated release of [14C]ACh release by SR 141716A was observed. Basal [14C]ACh release was unaffected by WIN 55212-2 or SR 141716A in either area. These results suggest that cannabinoid receptor activation can produce a strong inhibition of ACh release in the hippocampus. Furthermore, the potentiation of ACh release in the hippocampus by SR 141716A alone suggests either that this compound is an inverse agonist at cannabinoid receptors or it is antagonizing the actions of an endogenous ligand acting on these receptors.     

Modulation of CB1 cannabinoid receptor functions after a long-term exposure to agonist or inverse agonist in the Chinese hamster ovary cell expression system

We have investigated the adaptive changes of the human central cannabinoid receptor (CB1) stably expressed in Chinese hamster ovary cells (CHO-CB1), after agonist (CP 55,940) or selective CB1 inverse agonist (SR 141716) treatment. CB1 receptor density and affinity constant as measured by binding assays with both tritiated ligands remained essentially unchanged after varying period exposure of CHO-CB1 cells (from 30 min to 72 hr) to saturating concentrations of CP 55,940 or SR 141716. However, using a C-myc-tagged version of the CB1 receptor, FACS analysis and confocal microscopy studies on CB1 expression indicated that the agonist promoted a disappearance of cell surface receptor although inverse agonist increased its cell surface density. Taken together these results suggest that 1) agonist induces internalization of the receptor into a cellular compartment that would be still accessible to both the hydrophobic ligands CP 55,940 or SR 141716; 2) inverse-agonist promotes externalization of the receptor from an intracellular preexisting pool to the cell surface. In parallel, we also investigated the associated effects of CP 55,940 and SR 141716 on CB1 receptor-coupled second messengers. We showed that preexposure of cells to CP 55,940 induced a rapid desensitization of the CB1 to the agonist response. The ability of CP 55,940 to inhibit the forskolin-stimulated adenylyl cyclase and to activate the mitogen-activated protein kinase activity was dramatically reduced. By striking contrast, SR 141716 pretreatment of CHO-CB1 cells not only had no significant effect on the potency of CP 55,940 to inhibit the forskolin-stimulated adenylyl cyclase but also induced a significant enhancement of the CP 55,940 ability to stimulate the mitogen-activated protein kinase activity. These results suggest that the modulation of the number of cell surface receptor could lead to functional desensitization or sensitization of the CB1 receptors.     

Stimulation by DIF causes an increase of intracellular Ca2+ in Dictyostelium discoideum

Using fluorescence-activated cell sorting (FACS), we have studied the effect of the differentiation-inducing factor (DIF) on cellular Ca2+ in Dictyostelium discoideum. We have shown previously that freshly starved or postaggregation amoebae are heterogenous with respect to the amounts of cellular Ca2+ that they contain; the L or "low Ca2+" class exhibits a prespore tendency and the H or "high Ca2+" class exhibits a prestalk tendency. Upon adding DIF, within 2 min there is an approximately twofold increase in the relative fraction of amoebae falling in the H class. A major part of the increase is caused by Ca2+ influx from the extracellular medium. Therefore a rise in the level of cellular Ca2+ is an early step in the signal transduction pathway following stimulation by DIF. Also, in parallel with the cellular heterogeneity in respect of Ca2+ content, there is a heterogeneity in the response to DIF, which appears to be restricted to L cells.     

Relation of exocytotic release of gamma-aminobutyric acid to Ca2+ entry through Ca2+ channels or by reversal of the Na+/Ca2+ exchanger in synaptosomes

The specific inhibitor of the gamma-aminobutyric acid (GABA) carrier, NNC-711, (1-[(2-diphenylmethylene)amino]oxyethyl)- 1,2,5,6-tetrahydro-3-pyridine-carboxylic acid hydrochloride, blocks the Ca(2+)-independent release of [3H]GABA from rat brain synaptosomes induced by 50 mM K+ depolarization. Thus, in the presence of this inhibitor, it was possible to study the Ca(2+)-dependent release of [3H]GABA in the total absence of carrier-mediated release. Reversal of the Na+/Ca2+ exchanger was used to increase the intracellular free Ca2+ concentration ([Ca2+]i) to test whether an increase in [Ca2+]i alone is sufficient to induce exocytosis in the absence of depolarization. We found that the [Ca2+]i may rise to values above 400 nM, as a result of Na+/Ca2+ exchange, without inducing release of [3H]GABA, but subsequent K+ depolarization immediately induced [3H]GABA release. Thus, a rise of only a few nanomolar Ca2+ in the cytoplasm induced by 50 mM K+ depolarization, after loading the synaptosomes with Ca2+ by Na+/Ca2+ exchange, induced exocytotic [3H]GABA release, whereas the rise in cytoplasmic [Ca2+] caused by reversal of the Na+/Ca2+ exchanger was insufficient to induce exocytosis, although the value for [Ca2+]i attained was higher than that required for exocytosis induced by K+ depolarization. The voltage-dependent Ca2+ entry due to K+ depolarization, after maximal Ca2+ loading of the synaptosomes by Na+/Ca2+ exchange, and the consequent [3H]GABA release could be blocked by 50 microM verapamil. Although preloading the synaptosomes with Ca2+ by Na+/Ca2+ exchange did not cause [3H]GABA release under any conditions studied, the rise in cytoplasmic [Ca2+] due to Na+/Ca2+ exchange increased the sensitivity to external Ca2+ of the exocytotic release of [3H]GABA induced by subsequent K+ depolarization. Thus, our results show that the vesicular release of [3H]GABA is rather insensitive to bulk cytoplasmic [Ca2+] and are compatible with the view that GABA exocytosis is triggered very effectively by Ca2+ entry through Ca2+ channels near the active zones.     

Characterization and distribution of binding sites for [3H]-SR 141716A, a selective brain (CB1) cannabinoid receptor antagonist, in rodent brain

SR 141716A belongs to a new class of compounds (diarylpyrazole) that inhibits brain cannabinoid receptors (CB1) in vitro and in vivo. The present study showed that [3H]-SR 141716A binds with high affinity (Kd=0.61 +/- 0.06 nM) to a homogenous population of binding sites (Bmax=0.72 +/- 0.05 pmol/mg of protein) in rate whole brain (minus cerebellum) synaptosomes. This specific binding was displaced by known cannabinoid receptor ligands with the following rank order of potency SR 141716A > CP 55,940 > WIN 55212-2 = delta9-THC > anandamide. Apart from anandamide, all these compounds were found to interact competitively with the binding sites labeled by [3H]-SR 141716A. On the other hand, agents lacking affinity for cannabinoid receptors were unable to displace [3H]-SR 141716A from its binding sites (IC50 > 10 microM). In addition, the binding of [3H]-SR 141716A was insensitive to guanyl nucleotides. Regional rat brain distribution of CB1 cannabinoid receptors detected by [3H]-SR 141716A saturation binding and autoradiographic studies, showed that this distribution was very similar to that found for [3H]-CP 55,940. In vivo, the [3H]-SR 141716A binding was displaced by SR 141716A with ED50 values of 0.39 +/- 0.07 and 1.43 +/- 0.29 mg/kg following intraperitoneal and oral administration, respectively. Finally, the [3H]-SR 141716A binding sites remained significantly occupied for at least 12 hr following oral administration of 3 mg/kg SR 141716A. Taken together, these results suggest that SR 141716A in its tritiated form is a useful research tool for labeling brain cannabinoid receptors (CB1) in vitro and in vivo.     

A Ca(2+)-sensing receptor mutation causes hypoparathyroidism by increasing receptor sensitivity to Ca2+ and maximal signal transduction

This is a follow-up report of a 14 1/2 year-old boy with Laron syndrome, who received twice daily therapy with IGF-I 120 micrograms/kg for 5 years that resulted in a linear growth of 40 cm. Concomitantly he became very obese which is attributed to IGF-I action via the insulin receptors.     

Ca2+ channel beta3 subunit enhances voltage-dependent relief of G-protein inhibition induced by muscarinic receptor activation and Gbetagamma

The Ca2+ channel beta subunit has been shown to reduce the magnitude of G-protein inhibition of Ca2+ channels. However, neither the specificity of this action to different forms of G-protein inhibition nor the mechanism underlying this reduction in response is known. We have reported previously that coexpression of the Ca2+ channel beta3 subunit causes M2 muscarinic receptor-mediated inhibition of alpha1B Ca2+ currents to become more voltage-dependent. We report here that the beta3 subunit increases the rate of relief of inhibition produced by a depolarizing prepulse and also shifts the voltage dependency of this relief to more hyperpolarized voltages; these effects are likely to be responsible for the reduction of inhibitory response of alpha1B channels to G-protein-mediated inhibition seen after coexpression of the Ca2+ channel beta3 subunit. Additionally, the beta3 subunit alters the rate and voltage dependency of relief of the inhibition produced by coexpressed Gbeta1gamma1, in a manner similar to the changes it produces in relief of M2 receptor-induced inhibition. We conclude that the Ca2+ channel beta3 subunit reduces the magnitude of G-protein inhibition of alpha1B Ca2+ channels by enhancing the rate of dissociation of the G-protein betagamma subunit from the Ca2+ channel alpha1B subunit.     

Binding of an adenosine A1 receptor agonist and adenosine A1 receptor antagonist to sheep pineal membranes

The pineal organ of vertebrates produces melatonin and adenosine. In lower vertebrates, adenosine modulates melatonin production. We report herein that 2-chloro-cyclopentyl-[3H]-adenosine ([3H]CCPA: adenosine A1 receptor agonist) and [3H]-cyclopentyl-1,3-dipropylxanthine ([3H]DPCPX: adenosine A1 receptor antagonist), bind specifically to sheep pineal membranes. Binding of [3H]CCPA reached equilibrium at 90 min and dissociation revealed the presence of two components. Saturation analysis suggested the presence of a single population of binding sites (Kd = 1.67 +/- 0.06 nM, Bmax = 2386 fmol/mg protein). Binding was sensitive to GTP and GTPgammaS. Binding of [3H]DPCPX reached equilibrium at 60 min and dissociation was monophasic. Saturation analysis revealed a single population of binding sites (Kd = 5.8 +/- 1.12 nM, Bmax = 1116 fmol/mg protein). The specificity of the [3H]-analogues used and the rank order potency of the competitors tested in the competition experiments suggested the presence of A1 receptors. Future investigations are necessary to elucidate the significance of the differences observed between the binding properties of the adenosine A1 receptor agonist and adenosine A1 receptor antagonist.     

Cyclic ADP-ribose as an endogenous regulator of the non-skeletal type ryanodine receptor Ca2+ channel

The skeletal and cardiac isoforms of the ryanodine receptor Ca2+ channel (RyRC) constitute the Ca2+ release pathway in sarcoplasmic reticulum of skeletal and cardiac muscles, respectively. A direct mechanical and a Ca(2+)-triggered mechanism (Ca(2+)-induced Ca2+ release) have been respectively proposed to explain the in situ activation of Ca2+ release in skeletal and cardiac muscle. In non-muscle cells, however, where the RyRC also participates in Ca2+ signalling, the mechanism of RyRC activation is unknown. Cyclic adenosine 5'-diphosphoribose (cADPR), which is present in many mammalian tissues, has been reported to induce Ca2+ release from ryanodine-sensitive intracellular Ca2+ stores in sea urchin eggs. Here we provide evidence that cADPR directly activates the cardiac but not the skeletal isoform of the RyRC. This, together with results on sea urchin eggs, suggests that cADPR is an endogenous activator of the non-skeletal type of RyRC and may thus have a role similar to inositol 1,4,5-trisphosphate in Ca2+ signalling.     

Orchestration of neuronal migration by activity of ion channels, neurotransmitter receptors, and intracellular Ca2+ fluctuations

The real-time observation of cell movement in acute cerebellar slices reveals that granule cells alter their shape concomitantly with changes in the mode and rate of migration as they traverse different cortical layers. Although the origin of local environmental cues responsible for these position-specific changes in migratory behavior remains unclear, several signaling mechanisms involved in controlling granule cell movement have emerged. The onset of one such mechanism is marked by the expression of voltage-gated ion channels and neurotransmitter receptors in postmitotic cells prior to the initiation of their migration. Granule cells start their radial migration after the expression of N-type Ca2+ channels and the N-methyl-D-aspartate subtype of glutamate receptors on the plasmalemmal surface. Blockade of the channel or receptor activity significantly decreases the rate of cell movement, indicating that the activation of these membrane constituents provides an essential signal for the translocation of granule cells. Another signal that controls the rate of cell migration is embedded in the combined amplitude and frequency components of Ca2+ fluctuations in the somata of migrating granule cells. Interestingly, each phase of Ca2+ fluctuation controls a separate phase of saltatory movement in the granule cells: The cells move forward during the phase of transient Ca2+ elevation and remain stationary during the troughs. Consequently, the changes in the amplitude and frequency components of Ca2+ fluctuations directly affect granule cell movement: Reducing the amplitude or frequency of Ca2+ fluctuations slows down the speed of cell movement, while the enhancement of these components accelerates migration. These findings suggest that signaling molecules present in the local cellular milieu encountered on the migratory route control the shape and motility of granule cells by modifying Ca2+ fluctuations in the soma through the activation of specific ion channels and neurotransmitter receptors.     

Identification of the amino terminus of neuronal Ca2+ channel alpha1 subunits alpha1B and alpha1E as an essential determinant of G-protein modulation

We have examined the basis for G-protein modulation of the neuronal voltage-dependent calcium channels (VDCCs) alpha1E and alpha1B. A novel PCR product of alpha1E was isolated from rat brain. This contained an extended 5' DNA sequence and was subcloned onto the previously cloned isoform rbEII, giving rise to alpha1Elong whose N terminus was extended by 50 amino acids. VDCC alpha1 subunit constructs were co-expressed with the accessory alpha2-delta and beta2a subunits in Xenopus oocytes and mammalian (COS-7) cells. The alpha1Elong showed biophysical properties similar to those of rbEII; however, when G-protein modulation of expressed alpha1 subunits was induced by activation of co-expressed dopamine (D2) receptors with quinpirole (100 nM) in oocytes, or by co-transfection of Gbeta1gamma2 subunits in COS-7 cells, alpha1Elong, unlike alpha1E(rbEII), was found to be G-protein-modulated, in terms of both a slowing of activation kinetics and a reduction in current amplitude. However, alpha1Elong showed less modulation than alpha1B, and substitution of the alpha1E1-50 with the corresponding region of alpha1B1-55 produced a chimera alpha1bEEEE, with G-protein modulation intermediate between alpha1Elong and alpha1B. Furthermore, deletion of the N-terminal 1-55 sequence from alpha1B produced alpha1BDeltaN1-55, which could not be modulated, thus identifying the N-terminal domain as essential for G-protein modulation. Taken together with previous studies, these results indicate that the intracellular N terminus of alpha1E1-50 and alpha1B1-55 is likely to contribute to a multicomponent site, together with the intracellular I-II loop and/or the C-terminal tail, which are involved in Gbetagamma binding and/or in subsequent modulation of channel gating.     

Cytosolic Ca2+ acts by two separate pathways to modulate the supply of release-competent vesicles in chromaffin cells

Recovery from depletion of the readily releasable pool of vesicles (RRP) in adrenal chromaffin cells was studied at differing basal [Ca2+]i or following protein kinase C (PKC) activation by phorbol esters. Following depletion, the pool size was estimated at varied times from cell capacitance jumps in response to paired depolarizations. The experimentally observed RRP recovery time course and steady-state size could be predicted from the measured [Ca2+]i signal assuming a Michaelis-Menten-type regulation of the vesicle supply by Ca2+. An elevated recruitment activity was observed at increased [Ca2+]i even when protein kinase C was blocked, but maximum effects could be obtained only after stimulation of PKC by phorbol esters or by prolonged elevations in [Ca2+]i. We suggest that, in chromaffin cells, elevated cytosolic Ca2+ modulates exocytotic plasticity via PKC-dependent and -independent pathways.     

Evidence that [Phe1 psi(CH2-NH)Gly2]nociceptin-(1-13)-NH2, a peripheral ORL-1 receptor antagonist, acts as an agonist in the rat spinal cord

OBJECTIVE: To compare the characteristics of patients who did and did not respond to a request for information regarding their cryopreserved pre-embryos. DESIGN: Mail survey. SETTING: Academic-assisted reproductive technology program. PATIENT(S): One hundred thirty-six patients with cryopreserved pre-embryos. INTERVENTION(S): Patients were surveyed by first-class mail regarding their plans for their cryopreserved pre-embryos and their interest in embryo donation. MAIN OUTCOME MEASURE(S): Age, number of stored pre-embryos, and duration of storage of responders and nonresponders at 6 weeks after mailing. RESULT(S): Eighty-three patients (62%) did not respond to the survey. Compared with responders, nonresponders were significantly older at the time of embryo cryopreservation, had fewer pre-embryos cryopreserved, and had the pre-embryos cryopreserved for a longer duration. Five responders (9%) expressed an interest in embryo donation. Three patients requested disposal of pre-embryos. Sixteen surveys (12%) were returned as undeliverable. As a group, these patients had the fewest pre-embryos cryopreserved and had the longest duration of storage. CONCLUSION(S): A disturbing number of patients with cryopreserved pre-embryos ignored efforts by our program to maintain contact. Older patients with few cryopreserved pre-embryos may require special attention to avoid abandonment.     

Porcine hepatic response to sepsis and its amplification by an adrenergic receptor alpha1 agonist and a beta2 antagonist

CDK9 has been recently shown to have increased kinase activity in differentiated cells in culture and a differentiated tissue-specific expression in the developing mouse. In order to identify factors that contribute to CDK9's differentiation-specific function, we screened a mouse embryonic library in the yeast two-hybrid system and found a tumor necrosis factor signal transducer, TRAF2, to be an interacting protein. CDK9 interacts with a conserved domain in the TRAF-C region of TRAF2, a motif that is known to bind other kinases involved in TRAF-mediated signaling. Endogenous interaction between the two proteins appears to be specific to differentiated tissue. TRAF2-mediated signaling may incorporate additional kinases to signal cell survival in myotubes, a cell type that is severely affected in TRAF2 knockout mice.     

Regulation of mouse skeletal muscle L-type Ca2+ channel by activation of the insulin-like growth factor-1 receptor

We investigated the modulation of the skeletal muscle L-type Ca2+ channel/dihydropyridine receptor in response to insulin-like growth factor-1 receptor (IGF-1R) activation in single extensor digitorum longus muscle fibers from adult C57BL/6 mice. The L-type Ca2+ channel activity in its dual role as a voltage sensor and a selective Ca2+-conducting pore was recorded in voltage-clamp conditions. Peak Ca2+ current amplitude consistently increased after exposure to 20 ng/ml IGF-1 (EC50 = 5.6 +/- 1.8 nM). Peak IGF-1 effect on current amplitude at -20 mV was 210 +/- 18% of the control. Ca2+ current potentiation resulted from a shift in 13 mV of the Ca2+ current-voltage relationship toward more negative potentials. The IGF-1-induced facilitation of the Ca2+ current was not associated with an effect on charge movement amplitude and/or voltage distribution. These phenomena suggest that the L-type Ca2+ channel structures involved in voltage sensing are not involved in the response to the growth factor. The modulatory effect of IGF-1 on L-type Ca2+ channel was blocked by tyrosine kinase and PKC inhibitors, but not by a cAMP-dependent protein kinase inhibitor. IGF-1-dependent phosphorylation of the L-type Ca2+ channel alpha1 subunit was demonstrated by incorporation of [gamma-32P]ATP to monolayers of adult fast-twitch skeletal muscles. IGF-1 induced phosphorylation of a protein at the 165 kDa band, corresponding to the L-type Ca2+ channel alpha1 subunit. These results show that the activation of the IGF-1R facilitates skeletal muscle L-type Ca2+ channel activity via a PKC-dependent phosphorylation mechanism.     

Cloning and expression of a group IV cytosolic Ca2+-dependent phospholipase A2 from rat pancreatic islets. Comparison of the expressed activity with that of an islet group VI cytosolic Ca2+-independent phospholipase A2

Stimulation of pancreatic islets with glucose induces phospholipid hydrolysis and accumulation of nonesterified arachidonic acid, which may play signaling or effector roles in insulin secretion. Of enzymes that catalyze phospholipid hydrolysis, islet beta-cells express low molecular weight secretory phospholipases A2 (PLA2) and a Group VI, Ca2+-independent PLA2 (iPLA2). Previous studies indicate that islets also express a protein recognized by antibodies against a Group IV, cytosolic, Ca2+-dependent PLA2 (cPLA2). To further examine the possible expression of cPLA2 by islets, we screened a rat islet cDNA library with a probe that recognizes cPLA2 sequence, and isolated a full-length cPLA2 cDNA. The rat islet cPLA2-deduced amino acid sequence is 96% identical to those of human and mouse cPLA2. Transfection of COS-7 cells with cPLA2 cDNA in an expression vector induced expression of Ca2+-dependent PLA2 activity and of a protein recognized by anti-cPLA2 antibody. Comparison of recombinant islet cPLA2 and iPLA2 activities expressed in transfected COS-7 cells indicated that iPLA2 but not cPLA2 is stimulated by ATP. Both activities are similarly sensitive to inhibition by arachidonyltrifluoromethyl ketone, but iPLA2 is more effectively inhibited by a haloenol lactone suicide substrate than cPLA2. RT-PCR experiments with RNA from purified islet beta-cells and from an alpha-cell-enriched population prepared by fluorescence-activated cell-sorting indicated that cPLA2 mRNA is more abundant in the beta-cell population. Immunoblotting analyses indicate that islets express cPLA2-immunoreactive protein, and that interleukin-1 does not affect its expression. The cPLA2 is thus one of at least three classes of PLA2 enzymes with distinct properties expressed in beta-cells.