Differential Effect of High Extracellular Ca2+ on K+ and Cl− Conductances in Murine Osteoclasts

Effects of the extracellular Ca²⁺ concentration ([Ca²⁺] _o ) on whole cell membrane currents were examined in mouse osteoclastic cells generated from bone marrow/stromal cell coculture. The major resting conductance in the presence of 1 mm Ca²⁺ was mediated by a Ba²⁺-sensitive, inwardly rectifying K⁺ (IR_K) current. A rise in [Ca²⁺] _o (5–40 mm) inhibited the IR_K current and activated an 4,4′-diisothiocyano-2,2′-stilbenedisulfonate (DIDS)-sensitive, outwardly rectifying Cl⁻ (OR_Cl) current. The activation of the OR_Cl current developed slowly and needed higher [Ca²⁺] _o than that required to inhibit the IR_K current. The inhibition of the IR_K current consisted of two components, initial and subsequent late phases. The initial inhibition was not affected by intracellular application of guanosine 5′-O-(3-thiotriphosphate) (GTPγS) or guanosine 5′-O-(2-thiodiphosphate) (GDPβS). The late inhibition, however, was enhanced by GTPγS and attenuated by GDPβS, suggesting that GTP-binding proteins mediate this inhibition. The activation of the OR_Cl current was suppressed by pretreatment with pertussis toxin, but not potentiated by GTPγS. An increase in intracellular Ca²⁺ level neither reduced the IR_K current nor activated the OR_Cl current. Staurosporine, an inhibitor for protein kinase C, did not modulate the [Ca²⁺] _o -induced changes in the IR_K and OR_Cl conductances. These results suggest that high [Ca²⁺] _o had a dual action on the membrane conductance of osteoclasts, an inhibition of an IR_K conductance and an activation of an OR_Cl conductance. The two conductances modulated by [Ca²⁺] _o may be involved in different phases of bone resorption because they differed in Ca²⁺ sensitivity, temporal patterns of changes and regulatory mechanisms. Received: 28 May 1996/Revised: 28 January 1997     

Two inward K+ channels in the xylem parenchyma cells of barley roots are regulated by G-protein modulators through a membrane-delimited pathway

In order to study the mechanism and regulation of K⁺ resorption from the xylem by the cells that border the xylem vessels (the xylem parenchyma cells), K⁺ inward-rectifying channels (KIRCs) in the plasma membrane of xylem parenchyma cells from Hordeum vulgare L. cv. Apex were studied using the patch-clamp technique. In the inside-out configuration, three different types of K⁺ channel and a further K⁺ conductance could be identified. Two of these channels, named KIRC1 and KIRC2, were activated by guanosine 5′-[β,γ-imido]triphosphate (Gpp(NH)p; 150 μM), a non-hydrolyzable derivative of GTP, indicating that channel activity was up-regulated by G-proteins; modulation of channel activity occurred via a membrane-delimited pathway, since the effect could be demonstrated in cell-free patches. At 100 mM external K⁺, KIRC1 had a conductance of 8 pS. There was no effect of ATP on channel activity. Likewise, addition of 150 μM guanosine 5′-[β-thio]diphosphate (GDPβS) or adenosine 5′-[γ-thio]triphosphate (ATPγS) failed to activate KIRC1, indicating nucleotide specificity of the effect. A second K⁺ channel, activated by Gpp(NH)p (KIRC2) with gating properties clearly different from the first one was less frequently observed. Four different substates could be identified; the main level had a conductance of about 2 pS. Gating below the Nernst potential of K⁺ (E_K) was voltage-independent. The channel closed at potentials more positive than E_K. A third, hyperpolarization-activated K⁺ channel, KIRC3, with a low open probability was encountered in inside-out patches. It had a conductance of 45 pS in 100 mM K⁺. Channel activity was not affected by the addition of G-protein modulators. Moreover, slowly activating inward currents carried by K⁺ were recorded in several patches that are ascribed to a `subpicosiemens conductance'. Neither GDPβS nor Gpp(NH)p appeared to have an effect on the currents. Whole-cell measurements with these G-protein modulators included in the pipette solution were in general agreement with the results obtained on cell-free patches. A statistical evaluation revealed that time-dependent inward currents were larger when the G-protein activator Gpp(NH)p was included in the pipette medium compared to measurements with the inhibitor GDPβS. With the GTP analogue, an additional instantaneous component was elicited that was ascribed to KIRC2 activity. Data are discussed with respect to the putative role of G-proteins in conveying hormonal signals. Regulation by G-protein may either serve to fine-tune K⁺ uptake by xylem parenchyma cells or to initiate depolarization, followed by salt-efflux through depolarization-activated cation and anion channels. Received 11 October 1996 / Accepted: 21 April 1997     

Dynamic reorganization of the alkaline phosphatase-containing compartment during chemotactic peptide stimulation of human neutrophils imaged by backscattered electrons

Scanning electron microscopy (EM) and cytochemical techniques were used to examine the alkaline phosphatase-containing compartment in human neutrophils after stimulation with nanomolar concentrations of N-formylmethionyl-leucyl-phenylalanine (10^–8M fMLP). Alkaline phosphatase (AlkPase) activity was demonstrated with a lead-based metal capture cytochemical method. The reaction product was visualized with the backscattered electron imaging mode of scanning EM, and analyzed by electron probe X-ray microanalysis. Alkaline phosphatase activity was detected only in fMLP-stimulated neutrophils; unstimulated neutrophils displayed no activity. Stimulation of human neutrophils with 10^–8 M fMLP induced a time-dependent intracellular redistribution of irregular round or tubular granules containing alkaline phosphatase activity, as seen by backscattering. The intracellular redistribution of alkaline phosphatase activity was accompanied by increased cytochemical activity on the cell surface. The reaction product was localized preferentially on ridges and folds of polar neutrophils. Reorganization of the AlkPase-containing compartment correlated with changes induced by fMLP in cell shape, ie, membrane ruffling and front-tail polarity, as observed with the secondary electron image mode of scanning EM. These findings demonstrate the intracellular reorganization, increase, and asymmetric distribution of alkaline phosphatase activity on the plasma membrane of human neutrophils after stimulation by chemotactic peptides.     

Cyclic GMP modulates the expression of Gi protein and adenylyl cyclase signaling in vascular smooth muscle cells

We have recently shown that the nitric oxide (NO) donor, SNAP, decreased the expression of Giα proteins and associated functions in vascular smooth muscle cells. Because NO stimulates soluble guanylyl cyclase and increases the levels of guanosine 3′,5′-cyclic monophosphate (cGMP), the present studies were undertaken to investigate whether cGMP can also modulate the expression of Gi proteins and associated adenylyl cyclase signaling. A10 vascular smooth muscle cells (VSMCs) and primary cultured cells from aorta of Sprague Dawley rats were used for these studies. The cells were treated with 8-bromoguanosine 3′,5′-cyclic monophosphate (8Br-cGMP) for 24 h and the expression of Giα proteins was determined by immunobloting techniques. Adenylyl cyclase activity was determined by measuring [³²P]cAMP formation for [α-³²P]ATP. Treatment of cells with 8-Br-cGMP (0.5 mM) decreased the expression of Giα-2 and Giα-3 by about 30–45%, which was restored towards control levels by KT5823, an inhibitor of protein kinase G. On the other and hand, the levels of Gsα protein were not altered by this treatment. The decreased expression of Giα proteins by 8Br-cGMP treatment was reflected in decreased Gi functions. For example, the inhibition of forskolin (FSK)-stimulated adenylyl cyclase activity by low concentrations of GTPγS (receptor-independent Gi functions) was significantly decreased by 8Br-cGMP treatment. In addition, exposure of the cells to 8Br-cGMP also resulted in the attenuation of angiotensin (Ang) II- and C-ANP_4–23 (a ring-deleted analog of atrial natriuretic peptide [ANP]-mediated inhibition of adenylyl cyclase activity (receptor-dependent functions of Gi). On the other hand, Gsα-mediated stimulations of adenylyl cyclase by GTPγS, isoproterenol and FSK were significantly augmented in 8Br-cGMP-treated cells. These results indicated the 8Br-cGMP decreased the expression of Giα proteins and associated functions in VSMCs. From these studies, it can be suggested that 8Br-cGMP-induced decreased levels of Gi proteins and resultant increased levels of cAMP may be an additional mechanism through which cGMP regulates vascular tone and thereby blood pressure.     

G Protein-mediated Activation of a Nonspecific Cation Current in Cultured Rat Retinal Pigment Epithelial Cells

We used whole-cell patch-clamp recording techniques to investigate G protein-activated currents in cultured rat retinal pigment epithelial (RPE) cells. Using 140 mm KCl intracellular and 130 mm NaCl extracellular solutions, rat RPE cells possessed both inward and outward K⁺ currents. Upon addition of the nonhydrolyzable guanine triphosphate analogue, guanosine-5′-O-(3-thiophosphate) (GTPγS, 0.1 mm), to the recording electrode, a nonspecific cation (NSC) current was elicited. The NSC current had a mean reversal potential of +5.7 mV in 130 mm extracellular NaCl with Cs⁺-aspartate in the pipette, and was not affected by alterations in the extracellular Ca²⁺ or Cl⁻ concentration. The GTPγS-activated current was found to be permeable to several monovalent cations (K⁺, Na⁺, choline, TRIS, and NMDG). Addition of fluoroaluminate, an activator of large molecular weight heterotrimeric GTP-binding proteins (G proteins), to the intracellular recording solution activated the NSC current. The G protein involved was pertussis toxin (PTX)-sensitive, since GTPγS failed to activate the NSC current in cells pretreated with PTX. Further investigation of second messenger molecules suggested that activation of the NSC current was not affected by alterations in intracellular Ca²⁺ or ATP. From these results, we conclude that a G protein-regulated NSC current is present in rat RPE cells. Activation of the NSC current may sufficiently depolarize RPE cells to activate outward K⁺ currents. This would provide a mechanism by which these cells could rid themselves of accumulated K⁺. Received: 25 January 1996/Revised: 24 April 1996     

A novel intracellular compartment with unusual secretory properties in human neutrophils

Human neutrophils contain a novel intracellular compartment that is distinct from the previously characterized azurophil and specific granules. This compartment is distinguished by the presence of cytochemically detectable alkaline phosphatase activity. The alkaline phosphatase-containing compartments are short rod-shaped organelles that rapidly undergo a dramatic reorganization upon cell stimulation with either a chemoattractant or an active phorbol ester. Biochemical analysis shows that in unstimulated neutrophils the majority of the alkaline phosphatase activity is intracellular, but after stimulation essentially all of this activity becomes associated with the cell surface. The exocytotic pathway is unusual in that these small organelles fuse to form elongated tubular structures before their association with the plasmalemma.     

Suppressive effect of rebamipide, an antiulcer agent, against activation of human neutrophils exposed to formyl-methionyl-leucyl-phenylalanine

Rebamipide, an antiulcer agent, has been shown to be able to prevent gastric mucosal injury resulting in part from activation of neutrophils. The mechanism of its suppressive action, however, remains to be established. The present study aimed to determine the effect of rebamipide on activation of isolated human neutrophils and to identify the signal transduction pathway involved in its regulation. In unstimulated cells, alkaline phosphatase activity was found residing in short rod-shaped intracellular granules. Upon stimulation with a chemotactic peptide formyl-methionyl-leucyl-phenylalanine, the granules fused to form elongated tubular structures and spherical vacuoles. Rebamipide inhibited reorganization of alkaline phosphatase-containing granules along with upregulation of alkaline phosphatase activity and CD16, a marker of the granules. It also suppressed chemotaxis, an increase in intracellular calcium ion concentration, and NADPH oxidase activation in cells stimulated with formyl-methionyl-leucyl-phenylalanine. In contrast, the drug showed no inhibitory action toward upregulation of alkaline phosphatase activity and CD16, and activation of NADPH oxidase in cells stimulated with phorbol myristate acetate, an activator of protein kinase C. These findings demonstrate that rebamipide exerts a broad spectrum of suppressive actions toward biological functions of human neutrophils stimulated with formyl-methionyl-leucyl-phenylalanine, but not with phorbol myristate acetate, and suggest that the upstream point of protein kinase C is the signal transduction pathway involved in its regulation.     

Functional characteristics of TRPC4 channels expressed in HEK 293 cells

The classical type of transient receptor potential (TRPC) channel is a molecular candidate for Ca²⁺-permeable cation channels in mammalian cells. Because TRPC4 and TRPC5 belong to the same subfamily of TRPC, they have been assumed to have the same physiological properties. However, we found that TRPC4 had its own functional characteristics different from those of TRPC5. TRPC4 channels had no constitutive activity and were activated by muscarinic stimulation only when a muscarinic receptor was co-expressed with TRPC4 in human embryonic kidney (HEK) cells. Endogenous muscarinic receptor appeared not to interact with TRPC4. TPRC4 activation by GTPγS was not desensitized. TPRC4 activation by GTPγS was not inhibited by either Rho kinase inhibitor or MLCK inhibitor. TRPC4 was sensitive to external pH with pK _a of 7.3. Finally, TPRC4 activation by GTPγS was inhibited by the calmodulin inhibitor W-7. We conclude that TRPC4 and TRPC5 have different properties and their own physiological roles. These authors contributed equally to this work.     

Role of vesicle-associated membrane protein-2, through Q-soluble N-ethylmaleimide-sensitive factor attachment protein receptor/R-soluble N-ethylmaleimide-sensitive factor attachment protein receptor interaction,in the exocytosis of specific and tertiary granules of human neutrophils

We have examined the role of the R-soluble N-ethylmaleimide-sensitive factor attachment protein receptor (SNARE) synaptobrevin-2/vesicle-associated membrane protein (VAMP)-2 in neutrophil exocytosis. VAMP-2, localized in the membranes of specific and gelatinase-containing tertiary granules in resting human neutrophils, resulted translocated to the cell surface following neutrophil activation under experimental conditions that induced exocytosis of specific and tertiary granules. VAMP-2 was also found on the external membrane region of granules docking to the plasma membrane in activated neutrophils. Specific Abs against VAMP-2 inhibited Ca(2+) and GTP-gamma-S-induced exocytosis of CD66b-enriched specific and tertiary granules, but did not affect exocytosis of CD63-enriched azurophilic granules, in electropermeabilized neutrophils. Tetanus toxin disrupted VAMP-2 and inhibited exocytosis of tertiary and specific granules. Activation of neutrophils led to the interaction of VAMP-2 with the plasma membrane Q-SNARE syntaxin 4, and anti-syntaxin 4 Abs inhibited exocytosis of specific and tertiary granules in electropermeabilized neutrophils. Immunoelectron microscopy showed syntaxin 4 on the plasma membrane contacting with docked granules in activated neutrophils. These data indicate that VAMP-2 mediates exocytosis of specific and tertiary granules, and that Q-SNARE/R-SNARE complexes containing VAMP-2 and syntaxin 4 are involved in neutrophil exocytosis.     

Low molecular weight GTP-binding proteins in human neutrophil granule membranes

Degranulation of neutrophils involves the differential regulation of the exocytosis of at least two populations of granules. Low molecular weight GTP-binding proteins (LMW-GBPs) have been implicated in the regulation of vesicular traffic in the secretory pathways of several types of cells. In the present study we identify distinct subsets of LMW-GBPs associated with the membranes of neutrophil-specific and azurophilic granules. Ninety-four percent of total [35S]guanosine 5'-(3-O-thio)triphosphate (GTP gamma S) binding activity was equally distributed between the plasma membrane and cytosol with the remaining 6% localized in the granules. In contrast, the cytosol contained only 10% of the total GTPase activity while the specific granules accounted for 13%. [alpha-32P]GTP binding to proteins transferred to nitrocellulose revealed LMW-GBPs in all fractions except the azurophilic granules. The specific granules contained three out of four bands which were found in the plasma membrane; these ranged from 20 to 23 kDa and all were resistant to alkaline extraction. Photoaffinity labeling with [alpha-32P]8-azido-GTP in the presence of micromolar Al3+ identified proteins of 25 and 26 kDa unique to azurophilic granules; these could not be labeled with [alpha-32P]8-azido-ATP and could be extracted by acidic but not alkaline pH. Botulinum C3-mediated [32P]ADP-ribosylation identified proteins of 16, 20, and 24 kDa both in plasma membranes and those of specific granules. An anti-ras monoclonal antibody, 142-24E5, recognized a 20-kDa protein localized to the plasma and specific granule membranes which could not be extracted by alkaline pH, was not a substrate for botulinum C3 ADP-ribosyltransferase, and was translocated from specific granules to plasma membrane after exposure of neutrophils to phorbol myristate acetate. We conclude that neutrophil-specific and azurophilic granules contain distinct subsets of LMW-GBPs which are uniquely situated to regulate the differential exocytosis of these two compartments.     

α2A-Adrenoceptor-specific Stimulation of [35S]GTPγS Binding to Membrane Preparations of Rat Frontal Cortex

Functional activation of α_2A adrenergic receptors in the crude membranes from rat frontal cortex was studied by a [³⁵S]-guanosine 5′-O-(γ-thiotriphosphate) ([³⁵S]GTPγS) binding assay. α_2A agonists UK14304 and guanfacine decreased the ability of GDP to compete with [³⁵S]GTPγS binding to the membranes and 0.1 mM GDP was found to be optimal for the following functional experiments. However, even after careful optimization of experimental conditions the specificity of ligands for rat α₂ adrenoceptors were not sufficient, as agonists as well as antagonists became activators of other signal transduction systems before achieving their maximal effect in the α_2A-adrenergic system. Only using compromising concentration of agonist (up to 1 μM UK14304) and antagonist (up to 1 μM RS79948) to inhibit agonist’s effect, allowed us to filtrate out α_2A specific effect for characterization of signal transduction in rat frontal cortex membranes for the comparison efficacies of this system for different animals from behavioral experiments.     

Stimulation and conformational change of G<Subscript>o</Subscript>α induced by GAP-43

GAP-43 and G_o are peripheral membrane proteins enriched in neuronal growth cone. GAP-43 was highly purified from bovine cerebral cortex and myristoylated G_oα was highly purified from Escherichia coli cotransformed with pQE60 G_oα and pBB131 (NMT). GAP-43 stimulated GTPγS binding to G_oα and the stimulation effect was dependent on concentration of GAP-43. Protein-protein binding experiments using CaM-Sepharose affinity media revealed that G_oα GDP bound GAP-43 directly to form intermolecular complex. This interaction induced conformational change of G_oα. In the presence of GAP-43, fluorescence spectrum of G_oα GDP blue shifted 4 nm; fluorescence intensity increased 35.3% and apparent quenching constant (Ksv) increased from (1.1 ±0.22) ×10⁵ to (4.1±0.43) × 10⁵ (M⁻¹). However, no obvious changes of fluorescence spectra of G_oα GTPγS were observed in the absence or presence of GAP-43. Our results indicated that GAP-43 induced conformational change of G_oα GDP so as to accelerate GDP release and subsequent GTPγS binding, which activates G proteins to trigger signal transduction and amplification. These results provided insights into understanding the function of G proteins in coupling between receptors and effectors and the key role of GDP/GTP exchange mode in GTPase cycle.     

Guanine nucleotide-induced polymerization of actin in electropermeabilized human neutrophils

The effects of exogenous guanine nucleotides on the polymerization of actin in human neutrophils were tested in an electropermeabilized cell preparation. Close to 40% permeabilization was achieved with a single electric discharge as measured by nucleic acid staining with ethidium bromide or propidium iodide with minimal (less than 2%) release of the cytoplasmic marker lactate dehydrogenase. In addition, electropermeabilized neutrophils retained their capacity to produce superoxide anions and to sustain a polymerization of actin in response to surface-receptor dependent stimuli such as chemotactic factors. Electropermeabilization produced a rapid and transient permeabilization that allowed the entry of guanine nucleotides into the cells. GTP and, to a larger extent, its nonhydrolyzable analog guanosine 5'-O-2-thiotriphosphate (GTP[S]), induced a time- and concentration-dependent polymerization of actin, as determined by increased staining with 7-nitrobenz-2-oxa-1,3-diazolylphallacidin. The effects of the aforementioned guanine nucleotides were antagonized by GDP[S], but were insensitive to pertussis toxin. Cholera toxin potentiated to a small degree the amount of actin polymerization induced by GTP[S]. These results provided direct evidence for the involvement of GTP-binding proteins in the regulation of the organization of the cytoskeleton of neutrophils, an event that is of crucial importance to the performance of the defense-oriented functions of these cells.     

A Large-Conductance Chloride Channel in Pigmented Ciliary Epithelial Cells Activated by GTPγS

A large-conductance (or maxi-) chloride channel was identified in bovine pigmented ciliary epithelial (PCE) cells using inside-out excised patch clamp recording. The channel had a mean conductance of 293 pS when excised patches were bathed in symmetrical 130 mm NaCl although the conductance decreased to 209 pS when the solution bathing the cytoplasmic face of the patch contained only 33 mm NaCl. The channel was highly selective for chloride, with a P _Cl/P _Na= 24. A flickery, reversible block was produced by the diuretic stilbene 4-acetamido-4′-isothiocyanatostilbene-2,2′-disulfonic acid (SITS), while 4,4′-diisothiocyanatostilbene-2,2′-disulfonic acid (DIDS) produced a permanent block. The channel was rarely active in cell-attached patches and usually required several minutes of polarization before activity could be detected in excised patches, a process known as metagenesis. Once activated, the channel was voltage-dependent and was mainly open within the voltage range −30 to +30 mV closing when the membrane was polarized to larger values. GTPγS (100 μm) activated the channel with a latency of 170 sec when applied to the cytoplasmic face of patches. This activation was not reversible upon return to control solution within the duration of the experiment. We assess the available evidence and suggest a role for this channel in volume regulation. Received: 24 June 1996/Revised: 18 February 1997     

Induction of Exocytosis from Permeabilized Mast Cells by the Guanosine Triphosphatases Rac and Cdc42

We applied recombinant forms of the Rho-related small guanosine triphosphatases (GTPases) Rac2 and Cdc42/G25K to permeabilized mast cells to test their ability to regulate exocytotic secretion. Mast cells permeabilized with streptolysin-O leak soluble (cytosol) proteins over a period of 5 min and become refractory to stimulation by Ca²⁺ and guanosine triphosphate (GTP)γS over about 20–30 min. This loss of sensitivity is likely to be due to loss of key regulatory proteins that are normally tethered at intracellular locations. Exogenous proteins that retard this loss of sensitivity to stimulation may be similar, if not identical, to those secretory regulators that are lost. Recombinant Rac and Cdc42/G25K, preactivated by binding GTPγS, retard the loss of sensitivity (run-down) and, more importantly, enable secretion to be stimulated by Ca²⁺ alone. Investigation of the concentration dependence of each of these two GTPases applied individually to the permeabilized cells, and of Cdc42/G25K applied in the presence of an optimal concentration of Rac2, has provided evidence for a shared effector pathway and also a second effector pathway activated by Cdc42/G25K alone. Dominant negative mutant (N17) forms of Rac2 and Cdc42/G25K inhibit secretion induced by Ca²⁺ and GTPγS. Our data suggest that Rac2 and Cdc42 should be considered as candidates for G_E, GTPases that mediate exocytosis in cells of hematopoeitic origin.     

[35S]GTPγS binding: A tool to evaluate functional activity of a cloned opioid receptor transiently expressed in COS cells

In this paper we propose a powerful procedure to measure functional activation of the mouse δ-opioid receptor transiently expressed in mammalian cells. Receptor stimulation was assessed using a population of electroporated COS cells, transfected at a 50% efficiency. Under those conditions, agonist-promoted activation of the receptor was measured by [³⁵S]GTPγS binding. Both BW373U86, an alkaloid compound, and DADLE, a peptide agonist, elicited increase of specific [³⁵S]GTPγS binding representing 300% of basal level. Maximal activation was compared to that obtained for the cloned receptor stably expressed in CHO cells. Agonist efficacy was similar in both expressions systems, demonstrating the high sensitivity of the proposed method applied to transient expression. Finally dose-response curves were found highly reproducible across transfection experiments, opening the possibility for a direct comparison of distinct recombinant receptor preparations. This method represents a powerfull tool for the study of opioid signal transduction at the receptor level. It may also be extended to investigate signalling properties of other Gi/Go coupled receptors. Special issue dedicated to Dr. Eric J. Simon.     

A Ca2+-activated Whole-Cell Cl− Conductance In Human Placental Cytotrophoblast Cells Activated Via a G Protein

Whole-cell patch clamp experiments were performed on cultured human cytotrophoblast cells incubated for 24–48 hr after their isolation from term placentas. Cl⁻-selective currents were examined using K⁺-free solutions. Under nonstimulated conditions, most cells initially expressed only small background leak currents. However, inclusion of 0.2 mm GTPγS in the electrode solution caused activation of an outwardly rectifying conductance which showed marked time-dependent activation at depolarized potentials above +20 mV. Stimulation of this conductance by GTPγS was found to be Ca²⁺-dependent since GTPγS failed to activate currents when included in a Ca²⁺-free electrode solution. In addition, similar currents could be activated by increasing the [Ca²⁺] of the pipette solution to 500 nm. The Ca²⁺-activated conductance was judged to be Cl⁻-selective, since reversal potentials were predicted by Nernst equilibrium potentials for Cl⁻. This conductance could also be reversibly inhibited by addition of the anion channel blocker DIDS to the bath solution at a dose of 100 μm. Preliminary experiments indicated the presence of a second whole-cell anion conductance in human cytotrophoblast cells, which may be activated by cell swelling. Possible roles for the Ca²⁺-activated Cl⁻ conductance in human placental trophoblast are discussed. Received: 9 November 1995/Revised: 18 January 1996     

The Hormonal Control of Uterine Luminal Fluid Secretion and Absorption

GABA_A receptors composed of α, β and γ subunits display a significantly higher single-channel conductance than receptors comprised of only α and β subunits. The pore of GABA_A receptors is lined by the second transmembrane region from each of its five subunits and includes conserved threonines at the 6′, 10′ and 13′ positions. At the 2′ position, however, a polar residue is present in the γ subunit but not the α or β subunits. As residues at the 2′, 6′ and 10′ positions are exposed in the open channel and as such polar channel-lining residues may interact with permeant ions by substituting for water interactions, we compared both the single-channel conductance and the kinetic properties of wild-type α1β1 and α1β1γ2S receptors with two mutant receptors, αβγ(S2′A) and αβγ(S2′V). We found that the single-channel conductance of both mutant αβγ receptors was significantly decreased with respect to wild-type αβγ, with the presence of the larger valine side chain having the greatest effect. However, the conductance of the mutant αβγ receptors remained larger than wild-type αβ channels. This reduction in the conductance of mutant αβγ receptors was observed at depolarized potentials only (E_Cl = −1.8 mV), which revealed an asymmetry in the ion conduction pathway mediated by the γ2′ residue. The substitutions at the γ2′ serine residue also altered the gating properties of the channel in addition to the effects on the conductance with the open probability of the mutant channels being decreased while the mean open time increased. The data presented in this study show that residues at the 2′ position in M2 of the γ subunit affects both single-channel conductance and receptor kinetics.     

Insulin Action on Polyunsaturated Phosphatidic Acid Formation in Rat Brain: An “In Vitro” Model with Synaptic Endings from Cerebral Cortex and Hippocampus

The highly efficient formation of phosphatidic acid from exogenous 1-stearoyl-2-arachidonoyl-sn-glycerol (SAG) in rat brain synaptic nerve endings (synaptosomes) from cerebral cortex and hippocampus is reported. Phosphatidic acid synthesized from SAG or 1,2-dipalmitoyl-sn-glycerol (DPG) was 17.5 or 2.5 times higher, respectively, than from endogenous synaptosomal diacylglycerides. Insulin increased diacylglycerol kinase (DAGK) action on endogenous substrate in synaptic terminals from hippocampus and cerebral cortex by 199 and 97%, respectively. Insulin preferentially increased SAG phosphorylation from hippocampal membranes. In CC synaptosomes insulin increased phosphatidic acid (PA) synthesis from SAG by 100% with respect to controls. Genistein (a tyrosine kinase inhibitor) inhibited this stimulatory insulin effect. Okadaic acid or cyclosporine, used as Ser/Threo protein phosphatase inhibitors, failed to increase insulin effect on PA formation. GTPγS and particularly NaF were potent stimulators of PA formation from polyunsaturated diacylglycerol but failed to increase this phosphorylation when added after 5 min of insulin exposure. GTPγS and NaF increased phosphatidylinositol 4,5 bisphosphate (PIP2) labeling with respect to controls when SAG was present. On the contrary, they decreased polyphosphoinositide labeling with respect to controls in the presence of DPG. Our results indicate that a DAGK type 3 (DAGKε) which preferentially, but not selectively, utilizes 1-acyl-2-arachidonoyl-sn-glycerol and which could be associated with polyphosphoinositide resynthesis, participates in synaptic insulin signaling. GTPγS and NaF appear to be G protein activators related to insulin and the insulin receptor, both affecting the signaling mechanism that augments phosphatidic acid formation.     

Lysophosphatidylinositol Stimulates [<Superscript>35</Superscript>S]GTPγS Binding in the Rat Prefrontal Cortex and Hippocampus

Lysophosphatidylinositol (LPI) is a biologically active lipid that produces a number of responses in cultured cells, and has been suggested to have neuroprotective properties in vivo. Some of the actions of LPI are mediated by G-protein coupled receptors, but it is not known whether G-protein coupled receptor-mediated responses can be seen in intact brain tissue. In consequence, in the present study, we investigated autoradiographically whether LPI increased the [³⁵S]GTPγS binding level in brain tissue slices. In standard assay conditions, where as a positive control a robust response to cannabinoid receptor activation by the agonist ligand CP55,940 was seen, there was no increase in the autoradiographic density over basal produced by LPI. However, when the conditions were modified (incubation at 4°C rather than at 25°C, incubation time increased to 3 h, GDP concentration reduced from 2 to 0.1 mM), a significant increase in [³⁵S]GTPγS autoradiographic density in response to 10 μM LPI was seen in the prefrontal cortex, hippocampus, and cortex at the level of the hippocampus, although the degree of increase was small and very variable. No significant increases were seen in the hypothalamus or cerebellum. It is concluded that LPI, in the right conditions, can activate a sufficient number of G-proteins in the rat prefrontal cortex and hippocampus to produce a response in the [³⁵S]GTPγS autoradiographic assay of G-protein coupled receptor function.