Pertussis Toxin-insensitive G-protein Research Articles

Helix 8 Leu in the CB1 Cannabinoid Receptor Contributes to Selective Signal Transduction Mechanisms

The intracellular C-terminal helix 8 (H8) of the CB(1) cannabinoid receptor deviates from the highly conserved NPXXY(X)(5,6)F G-protein-coupled receptor motif, possessing a Leu instead of a Phe. We compared the signal transduction capabilities of CB(1) with those of an L7.60F mutation and an L7.60I mutation that mimics the CB(2) sequence. The two mutant receptors differed from wild type (WT) in their ability to regulate G-proteins in the [(35)S]guanosine 5'-3-O-(thio)triphosphate binding assay. The L7.60F receptor exhibited attenuated stimulation by agonists WIN-55,212-2 and CP-55,940 but not HU-210, whereas the L7.60I receptor exhibited impaired stimulation by all agonists tested as well as by the inverse agonist rimonabant. The mutants internalized more rapidly than WT receptors but could equally sequester G-proteins from the somatostatin receptor. Both the time course and maximal N-type Ca(2+) current inhibition by WIN-55,212-2 were reduced in the mutants. Reconstitution experiments with pertussis toxin-insensitive G-proteins revealed loss of coupling to Galpha(i3) but not Galpha(0A) in the L7.60I mutant, whereas the reduction in the time course for the L7.60F mutant was governed by Galpha(i3). Furthermore, Galpha(i3) but not Galpha(0A) enhanced basal facilitation ratio, suggesting that Galpha(i3) is responsible for CB(1) tonic activity. Co-immunoprecipitation studies revealed that both mutant receptors were associated with Galpha(i1) or Galpha(i2) but not with Galpha(i3). Molecular dynamics simulations of WT CB(1) receptor and each mutant in a 1-palmitoyl-2-oleoylphosphatidylcholine bilayer suggested that the packing of H8 is different in each. The hydrogen bonding patterns along the helix backbones of each H8 also are different, as are the geometries of the elbow region of H8 (R7.56(400)-K7.58(402)). This study demonstrates that the evolutionary modification to NPXXY(X)(5,6)L contributes to maximal activity of the CB(1) receptor and provides a molecular basis for the differential coupling observed with chemically different agonists.

Potentiation of nicotinic currents by bradykinin in the paratracheal ganglia neurons of rats

The effects of bradykinin on nicotine-induced responses were investigated in neurons dissociated from rat paratracheal ganglia using the nystatin-perforated patch-clamp recording technique. When bradykinin (10 − 9 to 10 − 8 M) was pretreated and then simultaneously applied with 10 − 5 M nicotine, bradykinin potentiated the nicotine-induced currents. The potentiation was mimicked by [Hyp 3]-bradykinin and inhibited by HOE-140, pertussis toxin, neomycin and U-73122, but not U-73433. These results suggest that bradykinin potentiates nicotinic currents via bradykinin B 2 receptor, pertussis toxin-sensitive G-protein and phospholipase C. Since bradykinin inhibits the M-current via bradykinin B 2 receptor and pertussis toxin-insensitive G-protein [Mochidome, T., Ishibashi, H., Takahama, K., 2001. Bradykinin activates airway parasympathetic ganglion neurons by inhibiting M-currents. Neuroscience 105, 785–791.], it seemed that bradykinin B 2 receptor activated two distinct signal transduction pathways in the paratracheal ganglia neurons. This effect of bradykinin might cause enhanced synaptic transmission in paratracheal ganglia neurons and contribute to the aggravation of pathological conditions of the lower airway via enhanced acetylcholine release from the postganglionic nerve terminals.

Evidence for a novel K(+) channel modulated by alpha(1A)-adrenoceptors in cardiac myocytes.

Accumulating evidence suggests that steady-state K(+) currents modulate excitability and action potential duration, particularly in cardiac cell types with relatively abbreviated action potential plateau phases. Despite representing potential drug targets, at present these currents and their modulation are comparatively poorly characterized. Therefore, we investigated the effects of phenylephrine [PE; an alpha(1)-adrenoceptor (alpha(1)-AR) agonist] on a sustained outward K(+) current in rat ventricular myocytes. Under K(+) current-selective conditions at 35 degrees C and whole-cell patch clamp, membrane depolarization elicited transient (I(t)) and steady-state (I(ss)) outward current components. PE (10 microM) significantly decreased I(ss) amplitude, without significant effect on I(t). Preferential modulation of I(ss) by PE was confirmed by intracellular application of the voltage-gated K(+) channel blocker tetraethylammonium, which largely inhibited I(t) without affecting the PE-sensitive current (I(ss,PE)). I(ss,PE) had the properties of an outwardly rectifying steady-state K(+)-selective conductance. Acidification of the external solution or externally applied BaCl(2) or quinidine strongly inhibited I(ss,PE). However, I(ss,PE) was not abolished by anandamide, ruthenium red, or zinc, inhibitors of TASK acid-sensitive background K(+) channels. Furthermore, the PE-sensitive current was partially inhibited by external administration of high concentrations of tetraethylammonium and 4-aminopyridine, which are voltage-gated K(+) channel-blockers. Power spectrum analysis of I(ss,PE) yielded a large unitary conductance of 78 pS. I(ss,PE) resulted from PE activation of the alpha(1A)-AR subtype, involved a pertussis toxin-insensitive G-protein, and was independent of cytosolic Ca(2+). These results collectively demonstrate that alpha(1A)-AR activation results in the inhibition of an outwardly rectifying steady-state K(+) current with properties distinct from previously characterized cardiac K(+) channels.

Signal transduction pathways involved in macrophage migration induced by peritoneal fluid chemotactic factors in stages I and II endometriosis

Objective: To explore the role of G-protein coupled signaling pathways in activation of macrophage migration in endometriosis stages I and II. Design: Case controlled study. Setting: University hospital. Patient(s): Fifteen patients undergoing laparoscopy for elective sterilization (n = 5) or for diagnosis of endometriosis stages I and II associated with infertility (n = 10). Intervention(s): Peritoneal fluid samples were collected during laparoscopy. Main Outcome Measure(s): Macrophage migration induced by peritoneal fluid from patients with endometriosis stages I and II (PF SI-II) and potential G-protein coupled receptors and second messengers involved in macrophage activation. Result(s): Potential G-protein coupled receptors and second messengers involved in macrophage activation were evaluated after incubation of U-937 cells differentiated into macrophages with inhibitors of phospholipase A and C, adenylate cyclase, and protein kinase A and C. Macrophage chemotactic activity induced by PF SI-II was inhibited in the presence of a phospholipase C and A 2 inhibitor (IC 50= 30 μM) and after treatment with myristoylated protein kinase C peptide inhibitor (50 nM). An increase in inositol phosphate (IP 3) was also observed in macrophages exposed to PF SI-II. Activation of multiple G-proteins in macrophages was examined after exposure of cells to PF SI-II in the presence and absence of Bordettela pertussis and cholera toxins. No effect on macrophage migration was observed. Conclusion(s): Macrophage chemotaxis induced by PF SI-II appears to involve activation of pertussis toxin-insensitive G-protein coupled receptors in macrophages. Our data suggest that these events lead to subsequent activation of phospholipases followed by generation of IP 3 and potential mobilization of intracellular Ca 2+. Subsequent phosphorylation of target proteins by protein kinase C may regulate the chemotactic responses. The adenylate cyclase pathway does not appear to play a role in this process.

The role of rat cytokine-induced neutrophil chemoattractants (CINCs) in inflammation

Rat cytokine-induced neutrophil chemoattractants (CINCs) are the members of the CXC chemokine family. Four neutrophil chemokines, CINC-1, CINC-2 alpha, CINC-2 beta and CINC-3, were purified from the conditioned medium of granulation-tissue culture. CINC-2 alpha and CINC-2 beta differ only in the sequence of three carboxy-terminal residues and are produced by alternative splicing. CINC-3 had neutrophil chemotactic activity similar to that of CINC-1 and CINC-2, but induced greater calcium mobilization than CINC-1 and CINC-2. CINC-1, -2 and -3 induced calcium flux in CXCR2-transfected HEK293 cells. In addition, anti-CXCR2 serum inhibited neutrophil chemotactic activities of the three types of CINCs almost completely. These results indicate that rat CXCR2 is a unique receptor for CINC-1, -2 and -3. CINCs induced calcium mobilization through pertussis toxin-insensitive G-protein but induced chemotaxis through pertussis toxin-sensitive G-protein. CINC-1/-2 and CINC-3 may stimulate both G-proteins with distinct efficiency. The concentration of CINC-1 increased transiently in rat air pouch/lipopolysaccharide inflammation, whereas the CINC-2 level increased linearly. The number of infiltrated cells increased up to 8 h. The increase in cell number was correlated with the total concentration of CINC-1 and CINC-2. Northern blot analyses and enzyme-linked immunosorbent assay showed that CINC expression was very low in rat macrophages without stimulation and increased after lipopolysaccharide stimulation. These data suggest that CINCs are expressed by inflammatory cells such as macrophages at a site of inflammation and play important roles in neutrophil infiltration.

Functional Specificity of Gαqand Gα11in the Cholinergic and Glutamatergic Modulation of Potassium Currents and Excitability in Hippocampal Neurons

In hippocampal and other cortical neurons, action potentials are followed by a slow afterhyperpolarization (sAHP) generated by the activation of small-conductance Ca(2+)-activated K(+) channels and controlling spike frequency adaptation. The corresponding current, the apamin-insensitive sI(AHP), is a well known target of modulation by different neurotransmitters, including acetylcholine (via M(3) receptors) and glutamate (via metabotropic glutamate receptor 5, mGluR(5)), in CA1 pyramidal neurons. The actions of muscarinic and mGluR agonists on sI(AHP) involve the activation of pertussis toxin-insensitive G-proteins. However, the pharmacological tools available so far did not permit the identification of the specific G-protein subtypes transducing the effects of M(3) and mGluR(5) on sI(AHP). In the present study, we used mice deficient in the Galpha(q) and Galpha(11) genes to investigate the specific role of these G-protein alpha subunits in the cholinergic and glutamatergic modulation of sI(AHP) in CA1 neurons. In mice lacking Galpha(q), the effects of muscarinic and glutamatergic agonists on sI(AHP) were nearly abolished, whereas beta-adrenergic agonists acting via Galpha(s) were still fully effective. Modulation of sI(AHP) by any of these agonists was instead unchanged in mice lacking Galpha(11). The additional depolarizing effects of muscarinic and glutamatergic agonists on CA1 neurons were preserved in mice lacking Galpha(q) or Galpha(11). Thus, Galpha(q), but not Galpha(11), mediates specifically the action of cholinergic and glutamatergic agonists on sI(AHP), without affecting the modulation of other currents. These results provide to our knowledge one of the first examples of the functional specificity of Galpha(q) and Galpha(11) in central neurons.

Regulation of Schwann Cell Morphology and Adhesion by Receptor-Mediated Lysophosphatidic Acid Signaling

In peripheral nerves, Schwann cells (SCs) form contacts with axons, other SCs, and extracellular matrix components that are critical for their migration, differentiation, and response to injury. Here, we report that lysophosphatidic acid (LPA), an extracellular signaling phospholipid, regulates the morphology and adhesion of cultured SCs. Treatment with LPA induces f-actin rearrangements resulting in a "wreath"-like structure, with actin loops bundled peripherally by short orthogonal filaments. The latter appear to anchor the SC to a laminin substrate, because they colocalize with the focal adhesion proteins, paxillin and vinculin. SCs also respond to LPA treatment by forming extensive cell-cell junctions containing N-cadherin, resulting in cell clustering. Pharmacological blocking experiments indicate that LPA-induced actin rearrangements and focal adhesion assembly involve Rho pathway activation via a pertussis toxin-insensitive G-protein. The transcript encoding LP(A1), the canonical G-protein-coupled receptor for LPA, is upregulated after sciatic nerve transection, and SCs cultured from lp(A1)-null mice exhibit greatly diminished morphological responses to LPA. Cultured SCs can release an LPA-like factor implicating SCs as a potential source of endogenous, signaling LPA. These data, together with the previous demonstration of LPA-mediated SC survival, implicate endogenous receptor-mediated LPA signaling in the control of SC development and function.

Membrane signalling and progesterone in female and male osteoblasts. I. Involvement of intracellular Ca2, inositol trisphosphate, and diacylglycerol, but not cAMP

Bone is a target tissue of progestins, but the mechanisms by which they act are still unclear. We examined the early (5–60 s) effects of progesterone and progesterone covalently bound to BSA (P-CMO BSA), which does not enter the cell, on the cytosolic free Ca2+ concentration ([Ca2+]i) and the formation of inositol 1,4,5 trisphosphate (InsP3) and diacylglycerol (DAG) in confluent female and male rat osteoblasts. P-CMO BSA like free progesterone increased [Ca2+]i via Ca2+ influx through L-type Ca2+ channels and Ca2+ mobilization from the endoplasmic reticulum. Both progestins increased InsP3 and DAG formation within 10 s, and the increase was blocked by phospholipase C inhibitors (neomycin and U-73122). Progesterone and P-CMO BSA mobilized calcium from the endoplasmic reticulum via the activation of a phospholipase C linked to a pertussis toxin-insensitive G-protein in both osteoblast types, and this process was controlled by protein kinase C. Neither progestin had any effect on cAMP formation in male and female osteoblasts. The membrane effects were not blocked by a progesterone nuclear antagonist. They were independent of the concentration of nuclear receptors and not linked to gender. Thus, progesterone appears to act in female and male rat osteoblasts via unconventional cell-surface receptors which belong to the class of membrane receptors coupled to phospholipase C via a pertussis toxin-insensitive G-protein. The bifurcating pathways leading to the formation of InsP3 and DAG may provide a certain flexibility in controlling cell responses, both by their nature and by their rates of formation and degradation. J. Cell. Biochem. 79:334–345, 2000. © 2000 Wiley-Liss, Inc.

Bradykinin, But Not Muscarinic, Inhibition of M-Current in Rat Sympathetic Ganglion Neurons Involves Phospholipase C-β4

Rat superior cervical ganglion (SCG) neurons express low-threshold noninactivating M-type potassium channels (I(K(M))), which can be inhibited by activation of M(1) muscarinic receptors (M(1) mAChR) and bradykinin (BK) B(2) receptors. Inhibition by the M(1) mAChR agonist oxotremorine methiodide (Oxo-M) is mediated, at least in part, by the pertussis toxin-insensitive G-protein Galpha(q) (Caulfield et al., 1994; Haley et al., 1998a), whereas BK inhibition involves Galpha(q) and/or Galpha(11) (Jones et al., 1995). Galpha(q) and Galpha(11) can stimulate phospholipase C-beta (PLC-beta), raising the possibility that PLC is involved in I(K(M)) inhibition by Oxo-M and BK. RT-PCR and antibody staining confirmed the presence of PLC-beta1, -beta2, -beta3, and -beta4 in rat SCG. We have tested the role of two PLC isoforms (PLC-beta1 and PLC-beta4) using antisense-expression constructs. Antisense constructs, consisting of the cytomegalovirus promoter driving antisense cRNA corresponding to the 3'-untranslated regions of PLC-beta1 and PLC-beta4, were injected into the nucleus of dissociated SCG neurons. Injected cells showed reduced antibody staining for the relevant PLC-beta isoform when compared to uninjected cells 48 hr later. BK inhibition of I(K(M)) was significantly reduced 48 hr after injection of the PLC-beta4, but not the PLC-beta1, antisense-encoding plasmid. Neither PLC-beta antisense altered M(1) mAChR inhibition by Oxo-M. These data support the conclusion of Cruzblanca et al. (1998) that BK, but not M(1) mAChR, inhibition of I(K(M)) involves PLC and extends this finding by indicating that PLC-beta4 is involved.

Activation of group I metabotropic glutamate receptors induces long-term depression at sensory synapses in superficial spinal dorsal horn

Low-frequency stimulation of primary afferent A δ-fibers can induce long-term depression of synaptic transmission in rat superficial spinal dorsal horn. Here, we have identified another form of long-term depression in superficial spinal dorsal horn neurons that is induced by specific group I but not group II metabotropic glutamate receptor (mGluR) agonists. Synaptic strength between A δ-fibers and dorsal horn neurons was examined by intracellular recordings in a spinal cord–dorsal root slice preparation from young rat. In the presence of bicuculline and strychnine, bath application of (1 S,3 R)-1-aminocyclopentane-1,3-dicarboxylic acid ((1 S,3 R)-ACPD) or the specific group I mGluR agonist ( S)-3,5-dihydroxyphenylglycine (( S)-3,5-DHPG) but not the specific group II mGluR agonist (2 S,2′ R,3′ R)-2-(2′,3′-dicarboxycyclopropyl)glycine (DCG-IV) for 20 min produced an acute and a long-term depression of synaptic strength. Bath application of the N-methyl- d-aspartate receptor antagonist d-2-amino-5-phosphonovaleric acid did not affect these depressions by (1 S,3 R)-ACPD. After pre-incubation of slices with pertussis toxin, a G-protein inhibitor, (1 S,3 R)-ACPD still induced acute and long-term depressions. The phospholipase C inhibitor U73122 stereoselectively blocked the induction of long-term depression without affecting acute synaptic inhibition. This study demonstrates that, in the spinal cord, direct activation of group I mGluRs that are coupled to phospholipase C through pertussis toxin-insensitive G-proteins induces a long-term depression of synaptic strength. This may be relevant to the processing of sensory information in the spinal cord, including nociception.

Muscarinic Stimulation of α1E Ca Channels Is Selectively Blocked by the Effector Antagonist Function of RGS2 and Phospholipase C-β1

Neuronal alpha1E Ca channel subunits are widely expressed in mammalian brain, where they are thought to form R-type Ca channels. Recent studies have demonstrated that R-type channels contribute to neurosecretion and dendritic Ca influx, but little is known concerning their modulation. Here we show that alpha1E channels are strongly stimulated, and only weakly inhibited, through M1 muscarinic acetylcholine receptors. Both forms of channel modulation are mediated by pertussis toxin-insensitive G-proteins. Channel stimulation is blocked by regulator of G-protein signaling 2 (RGS2) or the C-terminal region of phospholipase C-beta1 (PLCbeta1ct), which have been previously shown to function as GTPase-activating proteins for Galphaq. In contrast, RGS2 and PLCbeta1ct do not block inhibition of alpha1E through M1 receptors. Inhibition is prevented, however, by the C-terminal region of beta-adrenergic receptor kinase 1, which sequesters Gbetagamma dimers. Thus, stimulation of alpha1E is mediated by a pertussis toxin-insensitive Galpha subunit (e.g., Galphaq), whereas inhibition is mediated by Gbetagamma. The ability of RGS2 and PLCbeta1ct to selectively block stimulation indicates these proteins functioned primarily as effector antagonists. In support of this interpretation, RGS2 prevented stimulation of alpha1E with non-hydrolyzable guanosine 5'-0-(3-thiotriphosphate). We also report strong muscarinic stimulation of rbE-II, a variant alpha1E Ca channel that is insensitive to voltage-dependent inhibition. Our results predict that Galphaq-coupled receptors predominantly stimulate native R-type Ca channels. Receptor-mediated enhancement of R-type Ca currents may have important consequences for neurosecretion, dendritic excitability, gene expression, or other neuronal functions.

Voltage-dependent, pertussis toxin insensitive inhibition of calcium currents by histamine in bovine adrenal chromaffin cells.

Histamine is a known secretagogue in adrenal chromaffin cells. Activation of G-protein linked H(1) receptors stimulates phospholipase C, which generates inositol trisphosphate leading to release of intracellular calcium stores and stimulation of calcium influx through store operated and other channels. This calcium leads to the release of catecholamines. In chromaffin cells, the main physiological trigger for catecholamine release is calcium influx through voltage-gated calcium channels (I(Ca)). Therefore, these channels are important targets for the regulation of secretion. In particular N- and P/Q-type I(Ca) are subject to inhibition by transmitter/hormone receptor activation of heterotrimeric G-proteins. However, the direct effect of histamine on I(Ca) in chromaffin cells is unknown. This paper reports that histamine inhibited I(Ca) in cultured bovine adrenal chromaffin cells and this response was blocked by the H(1) antagonist mepyramine. With high levels of calcium buffering in the patch pipette solution (10 mM EGTA), histamine slowed the activation kinetics and inhibited the amplitude of I(Ca). A conditioning prepulse to +100 mV reversed the kinetic slowing and partially relieved the inhibition. These features are characteristic of a membrane delimited, voltage-dependent pathway which is thought to involve direct binding of G-protein betagamma subunits to the Ca channels. However, unlike virtually every other example of this type of inhibition, the response to histamine was not blocked by pretreating the cells with pertussis toxin (PTX). The voltage-dependent, PTX insensitive inhibition produced by histamine was modest compared with the PTX sensitive inhibition produced by ATP (28% vs. 53%). When histamine and ATP were applied concomitantly there was no additivity of the inhibition beyond that produced by ATP alone (even though the agonists appear to activate distinct G-proteins) suggesting that the inhibition produced by ATP is maximal. When experiments were carried out under conditions of low levels of calcium buffering in the patch pipette solution (0.1 mM EGTA), histamine inhibited I(Ca) in some cells using an entirely voltage insensitive pathway. We demonstrate that activation of PTX insensitive G-proteins (most likely Gq) by H(1) receptors inhibits I(Ca). This may represent a mechanism by which histamine exerts inhibitory (in addition to previously identified stimulatory) effects on catecholamine release.

Membrane signalling and progesterone in female and male osteoblasts. I. Involvement of intracellular Ca2+, inositol trisphosphate, and diacylglycerol, but not cAMP

Bone is a target tissue of progestins, but the mechanisms by which they act are still unclear. We examined the early (5-60 s) effects of progesterone and progesterone covalently bound to BSA (P-CMO BSA), which does not enter the cell, on the cytosolic free Ca(2+) concentration ([Ca(2+)]i) and the formation of inositol 1,4,5 trisphosphate (InsP3) and diacylglycerol (DAG) in confluent female and male rat osteoblasts. P-CMO BSA like free progesterone increased [Ca(2+)]i via Ca(2+) influx through L-type Ca(2+) channels and Ca(2+) mobilization from the endoplasmic reticulum. Both progestins increased InsP(3) and DAG formation within 10 s, and the increase was blocked by phospholipase C inhibitors (neomycin and U-73122). Progesterone and P-CMO BSA mobilized calcium from the endoplasmic reticulum via the activation of a phospholipase C linked to a pertussis toxin-insensitive G-protein in both osteoblast types, and this process was controlled by protein kinase C. Neither progestin had any effect on cAMP formation in male and female osteoblasts. The membrane effects were not blocked by a progesterone nuclear antagonist. They were independent of the concentration of nuclear receptors and not linked to gender. Thus, progesterone appears to act in female and male rat osteoblasts via unconventional cell-surface receptors which belong to the class of membrane receptors coupled to phospholipase C via a pertussis toxin-insensitive G-protein. The bifurcating pathways leading to the formation of InsP(3) and DAG may provide a certain flexibility in controlling cell responses, both by their nature and by their rates of formation and degradation.

Membrane signaling and progesterone in female and male osteoblasts. II. Direct involvement of G alpha q/11 coupled to PLC-beta 1 and PLC-beta 3.

We have shown that progesterone (10 pM-10 nM) and progesterone covalently bound to bovine serum albumin (P-CMO BSA; 100 pM-1 microM) rapidly increased (within 5 s) the cytosolic free Ca(2+) concentration and inositol 1,4,5 trisphosphate (InsP(3)) formation in confluent female and male rat osteoblasts via a pertussis toxin-insensitive G-protein. The activation of G-proteins coupled to effectors such as phospholipase C (PLC) is an early event in the signal transduction pathway leading to InsP(3) formation. We used antibodies against the various PLC isoforms to show that only PLC-beta1 and PLC-beta 3 were involved in the Ca(2+) mobilization and InsP(3) formation induced by both progestins in female and male osteoblasts, whereas PLC-beta 2, PLC-gamma 1, and PLC-gamma 2 were not. We also used antibodies against the subunits of heterotrimeric G-proteins to show that the activation of PLC-beta 1 and PLC-beta 3 by both progestins involved the G alpha q/11 subunit, which was insensitive to pertussis toxin, whereas G alpha i, G alpha s, and G beta gamma subunits were not. The membrane effects were independent of the concentration of nuclear progesterone receptor, because the concentration of nuclear progesterone receptors was lower in male than in female osteoblasts. These data suggest that progesterone and P-CMO BSA, which does not enter the cell, directly activate G-protein leading to the very rapid formation of second messengers without involving the nuclear receptor.

Pharmacological and Functional Characterization of Bradykinin Receptors in Rat Cultured Vascular Smooth Muscle Cells

The pharmacological properties of bradykinin receptors were characterized in rat cultured vascular smooth muscle cells (VSMCs) using [ 3H]-bradykinin as a ligand. Analysis of binding isotherms gave an apparent equilibrium dissociation constant ( K D) of 1.2 ± 0.2 nM and a maximum receptor density ( B max) of 47.3 ± 4.4 fmol/mg protein. The specific binding of [ 3H]-bradykinin to VSMCs was inhibited by the B 2 receptor-selective agonists (bradykinin and kallidin) and antagonists ([D-Arg 0, Hyp 3, Thi 5, D-Tic 7, Oic 8]-bradykinin (Hoe 140) and [D-Arg 0, Hyp 3, Thi 5,8, D-Phe 7]-bradykinin) with an order of potency as kallidin = bradykinin = Hoe 140 > [D-Arg 0, Hyp 3, Thi 5,8, D-Phe 7]-bradykinin, but not by a B 1 receptor-selective agonist (des-Arg 9-bradykinin) and antagonist ([Leu 8, des-Arg 9]-bradykinin). Stimulation of VSMCs by bradykinin produced a concentration-dependent inositol phosphate (IP) accumulation, and initial transient peak of [Ca 2+] i with half-maximal responses (pEC 50) were 7.53 and 7.69, respectively. B 2 receptor-selective antagonists (Hoe 140 and [D-Arg 0, Hyp 3, Thi 5,8, D-Phe 7]-bradykinin) significantly antagonized the bradykinin-induced responses with pK B values of 8.3–8.7 and 7.2–7.9, respectively. Pretreatment of VSMCs with pertussis toxin (100 ng/ml, 24 h) did not alter the bradykinin-induced inositol phosphate accumulation and [Ca 2+] i changes in VSMCs. Removal of external Ca 2+ led to a significant attenuation of responses induced by bradykinin. Influx of external Ca 2+ was required for the bradykinin-induced responses, since Ca 2+-channel blockers, nifedipine, verapamil, and Ni 2+, partially inhibited the bradykinin-induced IP accumulation and Ca 2+ mobilization. These results demonstrate that bradykinin stimulates phosphoinositide hydrolysis and Ca 2+ mobilization via a pertussis toxin-insensitive G-protein in rat VSMCs. Bradykinin B 2 receptors may be predominantly mediating IP accumulation and subsequently induction of Ca 2+ mobilization may function as the transducing mechanism for bradykinin-stimulated contraction of vascular smooth muscle.

Functional Characterization of α1-Adrenergic Receptors in Experimental Vein Grafts

Background.Studies on the pharmacology of the smooth muscle cells in vein bypass grafts suggest that the function of G-proteins and adrenergic receptors is altered. This study examines the α-adrenergic responsiveness of smooth muscle cells in vein bypass grafts as compared with those in the common carotid arteries and external jugular veins.Methods.New Zealand White rabbits received jugular vein interposition bypass grafts of the common carotid. Vessel segments of the vein bypass grafts harvested after 28 days, common carotid arteries, and external jugular veins were sectioned into 5-mm rings (four per vessel) for studies of isometric tension in response to phenylephrine (10−10to 10−4M) alone and in the presence of prazosin, an α1-adrenergic antagonist; WB4101 and 5-methylurapidil (5-MU), α1Aantagonists; chloroethylclonidine (CEC); an α1Bantagonist; or the Gi/oG-protein inhibitor pertussis toxin (PTx).Results.All vessels had prazosin-sensitive responses. The jugular veins appear to have functional α1Areceptors (WB4101 and 5-MU sensitive, CEC insensitive) which are associated with pertussis toxin-sensitive G-proteins. Carotid arteries appear to have atypical α1receptors (WB4101 and 5-MU insensitive, CEC insensitive) associated with pertussis toxin-insensitive G-proteins. Vein grafts appear to have functional α1Breceptors (WB4101 and 5-MU insensitive, CEC sensitive) which are associated with pertussis toxin-insensitive G-proteins.Conclusions.These results show that placement of a vein into the arterial circulation induces a change in α1-adrenergic receptor subtypes (α1Ato α1B) and in the G-protein coupling of the receptors (PTx sensitive to PTx insensitive), reflecting a signficant phenotypic change in smooth muscle cell signal transduction.

Angiotensin AT 1 receptors in Clone 9 rat liver cells: Ca 2+ signaling and c- fos expression

In C9 (Clone 9) liver cells, angiotensin II increased the intracellular Ca 2+ content, inositol phosphate production and c- fos mRNA expression. Other angiotensins were also active with the order of potency being angiotensin II=angiotensin III≫angiotensin I>angiotensin IV. Losartan, but not PD 123177 (1-(4-amino-3-methyl)-5-diphenylacetyl-4,5,6,7-tetrahydro-1 H-imidazo [4,5 c]pyridine-6-carboxylic acid), blocked the effects of angiotensin II. Pertussis toxin did not alter these actions of angiotensin II. These data indicate that the effects were mediated through angiotensin AT 1 receptors involving pertussis toxin-insensitive G-proteins. Phorbol myristate acetate was also able to increase c- fos mRNA expression. The action of angiotensin II was consistently greater than that of the active phorbol ester. Staurosporine but not genistein inhibited this effect of angiotensin II. Angiotensin II- and phorbol myristate acetate-induced proto-oncogene mRNA expression was attenuated in cells incubated overnight with the active phorbol ester, which suggests a major role of protein kinase C.

The α Subunit of GqContributes to Muscarinic Inhibition of the M-Type Potassium Current in Sympathetic Neurons

Rat superior cervical ganglion (SCG) neurons express low-threshold noninactivating M-type potassium channels (IK(M)), which can be inhibited by activation of M1 muscarinic receptors. This inhibition occurs via pertussis toxin-insensitive G-proteins belonging to the Galphaq family (Caulfield et al., 1994 ). We have used DNA plasmids encoding antisense sequences against the 3' untranslated regions of Galpha subunits (antisense plasmids) to investigate the specific G-protein subunits involved in muscarinic inhibition of IK(M). These antisense plasmids specifically reduced levels of the target G-protein 48 hr after intranuclear injection. In cells depleted of Galphaq, muscarinic inhibition of IK(M) was attenuated compared both with uninjected neurons and with neurons injected with an inappropriate GalphaoA antisense plasmid. In contrast, depletion of Galpha11 protein did not alter IK(M) inhibition. To determine whether the alpha or beta gamma subunits of the G-protein mediated this inhibition, we have overexpressed the C terminus of beta adrenergic receptor kinase 1 (betaARK1), which binds free beta gamma subunits. betaARK1 did not reduce muscarinic inhibition of IK(M) at a concentration of plasmid that can reduce beta gamma-mediated inhibition of calcium current (). Also, expression of beta1gamma2 dimers did not alter the IK(M) density in SCG neurons. In contrast, IK(M) was virtually abolished in cells expressing GTPase-deficient, constitutively active forms of Galphaq and Galpha11. These data suggest that Galphaq is the principal mediator of muscarinic IK(M) inhibition in rat SCG neurons and that this more likely results from an effect of the alpha subunit than the beta gamma subunits of the Gq heterotrimer.

Thapsigargin and receptor-mediated activation of Drosophila TRPL channels stably expressed in a Drosophila S2 cell line

The Drosophila melanogaster genes, transient receptor potential ( trp) and transient receptor potential-like ( trpl) encode putative plasma membrane cation channels TRP and TRPL, respectively. We have stably co-expressed Drosophila TRPL with a Drosophila muscarinic acetylcholine receptor (DM1) in a Drosophila cell line (S2 cells). Basal Ca 2+ levels measured using Fura-2/AM in unstimulated S2-DM1-TRPL cells were low and indistinguishable from untransfected cells, indicating that the TRPL channels were not constitutively active in this expression system. Activation of DM1 receptor in S2-DM1-TRPL cells by 100 μM carbamylcholine induced Ca 2+ release from an intracellular Ca 2+ pool followed by a Gd 3+-insensitive Ca 2+ influx. Pretreatment of S2-DM 1-TRPL cells with 10 μM atropine abolished Gd 3+-insensitive Ca 2+ influx triggered by carbamylcholine, but the response was not blocked by prior incubation with pertussis toxin. TRPL channels could also be reliably activated by bath application of 1 μM thapsigargin for 10 min or 100 nM thapsigargin for 60 min in Ca 2+-free solution. In some cells, TRPL channels activated by thapsigargin could further be activated by carbamylcholine. The findings suggest that, when stably expressed in the S2 cell line, TRPL may be regulated by two distinct mechanisms: (i) store depletion; and (ii) stimulation of DM1 receptor via pertussis-toxin insensitive G-protein (or the subsequent activation of PLC), but without further requirement for Ca 2+ release.

Differential localization of the mRNAs for the pertussis toxin insensitive G-protein alpha sub-units G q, G 11, and G z in the rat brain, and regulation of their expression after striatal deafferentation

The corticostriatal pathway is among the largest glutamatergic pathways in the brain, and of particular interest to the study of glutamatergic transmission. The metabotropic glutamate receptors (mGluRs) couple the actions of glutamate to intracellular second messenger systems through G-proteins. The most prominent of the mGluRs present in the target of this pathway, the striatum, is mGluR5. The identity of the G-proteins mediating the actions of mGluR5 are unknown, but the receptor is linked to stimulation of phosphoinositide (PI) turnover and largely resistant to the effects of pertussis toxin, which inhibits some G-proteins. We used in situ hybridization to examine the expression and regulation of three pertussis toxin insensitive G-protein α sub-units: G q, G 11, and G z. We found that these mRNAs are differentially distributed in the rat brain, but all three are expressed by striatal neurons. After glutamatergic deafferentation of the striatum by decortication, there is a modest upregulation of G 11 mRNA, while expression of G q and G z are unchanged. Following dopaminergic deafferentation, expression of G q, G 11, and G z are not altered, although expression of the pertussis-sensitive sub-unit G o is increased. Our data suggests that G z, G q, and G 11 are each expressed by striatal neurons, and therefore may be involved in mediating the actions of mGluR5 in these cells. After decortication G 11 is upregulated, but the magnitude of this effect is small, and alone seems insufficient to account for the marked biochemical supersensitivity of glutamate-stimulated PI turnover which is observed.