Neuronal Platelet-Activating Factor Receptor Signal Transduction Involves a Pertussis Toxin-Sensitive G-Protein

In most nonneural systems, platelet-activating factor (PAF) receptor effects are mediated by G-proteins that are often pertussis toxin-sensitive. The activation of pertussis toxin-sensitive G-proteins linked to PAF receptors results in the mobilization of intracellular calcium, at least in part, through the second messenger inositol triphosphate. We have sought to determine if a pertussis toxin-sensitive G-protein is involved in the PAF receptor-mediated phenomena of growth cone collapse and of synaptic enhancement in primary neuronal culture. Using infrared differential interference contrast microscopy and patch-clamp recording techniques, pertussis toxin, but not the inactive B oligomer of the toxin, was found to block both the growth cone collapse and the enhanced synaptic release of excitatory transmitter induced by a nonhydrolyzable PAF receptor agonist, making it likely that G_o, G_q, or G_i is the G-protein transducer of PAF receptors in primary neurons. We believe that PAF acts directly on neuronal receptors, which are linked to pertussis toxin-sensitive G-proteins, on the tips of developing neurites, and on presynaptic nerve terminals, leading to growth cone collapse and enhanced synaptic release of transmitter.     

Cerebral Cortex Gsα Protein Levels and Forskolin-Stimulated Cyclic AMP Formation Are Increased in Bipolar Affective Disorder

Abstract: Experimental animal and peripheral blood cell studies point to guanine nucleotide regulatory (G) protein disturbances in bipolar affective disorder. We have previously reported elevated prefrontal cortex G_sα protein in bipolar affective disorder and have now extended these preliminary observations in a larger number of subjects, assessing the brain regional specificity of these changes in greater detail, determining the functional biochemical correlates of such changes, and evaluating their diagnostic specificity. Membrane G protein (G_sα, G_iα, G_oα, and Gβ) immunoreactivities were estimated by western blotting in postmortem brain regions obtained from 10 patients with a DSMIII-R diagnosis of bipolar affective disorder and 10 nonpsychiatric controls matched on the basis of age, postmortem delay, and brain pH. To examine whether there were functional correlates to the observed elevated G_sα levels, basal and GTPγS-and forskolin-stimulated cyclic AMP production was determined in the same brain regions. Compared with controls, G_sα (52-kDa species) immunoreactivity was significantly (p < 0.05) elevated in prefrontal (+36%), temporal (+65%), and occipital (+96%) cortex but not in hippocampus (+28%), thalamus (-23%), or cerebellum (+21%). In contrast, no significant differences were found in the other G protein subunits (G_iα, G_oα, Gβ) measured in these regions. Forskolin-stimulated cyclic AMP production was significantly increased in temporal (+31%) and occipital (+96%) cortex but not in other regions. No significant differences were apparent in basal or GTPγS-stimulated cyclic AMP production. A significant correlation (r= 0.60, p < 0.001) was observed between forskolin-stimulated cyclic AMP formation and G_sα (52 kDa) immunoreactivity when examined across these cortical regions. The observed increase in G_sα may be specific to bipolar disorders as no significant differences were detected in G_sα levels in temporal cortex from patients with either schizophrenia (n = 7) or Alzheimer's disease (n = 7). In summary, the present study confirms and extends our earlier findings and supports the notion that increased G_sα levels and possibly G_sα-adenylyl cyclase-mediated signal transduction are relevant to the pathophysiology of bipolar affective disorder.     

Distribution of the α-subunit of the Guanine Nucleotide-binding Protein Gi2 and its Comparison to Gαo

Abstract

Site specific antisera against a synthetic peptide corresponding to the sequence 3–17 of Gαi2 have been raised and the specificity examined using purified homogeneous G_o, G_i2 and G_i containing a 41 kDa α-subunit. The distribution of Gα_i2 was investigated in plasma membranes from different tissues and cells and compared to the distribution of Gα_o and other pertussis toxin sensitive Gα. Considerable amounts of Gα_io were found in endocrine tissue especially in membranes from the adrenal and thyroid, in leucocytes and platelets where it constitutes the major, if not only, pertussis toxin-sensitive Gα, as well as in some cell lines (C6, NG 108–15, S49 cyc^?); erythrocytes contained a 41 kDa Gα_i which was different from Gα_o. Gα_o was present abundantly in nervous tissue, adrenal medulla and cortex but also found in low amounts in other membranes except for lung, liver and blood cells. Subcellular fractionaltion of cardiac ventricular muscle demonstrated the presence of Gα_o and low amounts of Gα_i2 in sarcolemma, but only 41kDa Gα_i was present in sarcoplasmic reticulum. The importance of the distinct distribution in terms of signal transduction is discussed.     

Subcellular localization of G-proteins in primary-cultured mouse preadipocytes and adipocytes

The subcellular localization of G_sα, G_iα1&2, G_iα3, and Gβ was studied in primary-cultured undifferentiated and differentiated, lipid replete, adipose cells. The results show a distinct distribution for each of these G-proteins and differences between differentiated and undifferentiated cells. All the G-proteins examined had a cytoplasmic localization; only G_iα1 and 2 showed a significant colocalization with the plasma membrane and this only in differentiated cells. Most studies using cells in culture have reported an intracellular localization for G-proteins, whereas in tissue sections the localization has been reported to be largely with the plasma membrane, with some intracellular localization. The results suggest that the cell-cell interactions or the specific geometry imposed by culture conditions favor the intracellular compared to peripheral localization of G-proteins. Alternately, the posttranslational modifications necessary for G-protein insertion in the plasma membrane may be deficient in cultured cells. J. Cell. Biochem. 65:259–266. © 1997 Wiley-Liss, Inc.     

Pertussis toxin-sensitive G protein that supports vitellogenin uptake by promoting patency

Ovarian follicles of Hyalophora cecropia stopped accumulating [³⁵S]vitellogenin when incubated in pertussis toxin, a G_i protein inactivator. At a cellular level, the responses to pertussis toxin resembled those described earlier to cell-permeant analogs of cyclic AMP. They included accelerated ³⁶Cl^– exchange, ⁸⁶Rb⁺ uptake, and follicle cell swelling, which in turn resulted in a loss of epithelial patency. A 34% rise in follicular cAMP content accompanied these changes. In particulate fractions of follicle homogenates, pertussis toxin catalyzed the ADP-ribosylation of a polypeptide that resolved at 39 kDa in SDS-PAGE; rabbit antibodies to a C-terminal decapeptide common to 39 kDa mammalian G_iα-3 and G_oα were bound in immunoblots at this same location. The findings suggest that a pertussis toxin-sensitive Gα facilitates epithelial patency during vitellogenesis by suppressing cAMP levels. When follicles are released from this restraint, either experimentally with pertussis toxin or by progressing to the next phase in their normal program of development, cAMP levels rise and vitellogenesis terminates. Arch. Insect Biochem. Physiol. 39:36–45, 1998. © 1998 Wiley-Liss, Inc.     

Immunoprecipitation of a pertussis toxin substrate of the Go family from rat islets of Langerhans

Rat islets express a pertussis toxin sensitive G-protein involved in receptor-mediated inhibition of insulin secretion. This has been assumed previously to represent “G_i” which couples inhibitory receptors to adenylate cyclase. Incubation of islet G-proteins with³²P-NAD and pertussis toxin resulted in the labelling of a band of molecular weight 40,000. This band was very broad and did not allow resolution of individual components. Incubation of the radiolabelled proteins with an anti-G_o antiserum resulted in specific immunoprecipitation of a³²P-labelled band. These results demonstrate that the complement of pertussis toxin sensitive G-proteins in rat islets includes G_o.     

Guanine nucleotide regulatory protein alterations in young Milan hypertensive strain rats

Vascular smooth muscle cell membranes from prehypertensive rats of the Milan hypertensive strain (MHS) were used to examine adenylyl cyclase activity and its regulation by guanine nucleotide regulatory proteins (G-proteins). Basal adenylyl cyclase activity was similar in MHS and Milan normontensive strain (MNS) membranes. Forsokolin (10^?4 M) produced a significantly greater stimulatory response in MHS membranes, but this was not observed with NaF (10^?2 M). Isoporterenol (10^?4 M) caused a significantly decreased stimulation of adenylyl cyclase activity in MHS membranes, while prostaglandin E₁ (10^?5 M) produced similar responses in the two strains. G_i function and GTP responses, as observed by biphasic effects of GTP on isoproterenol-stimulated membranes, were similar in both strains. The levels of G_i2α and G_qα/G₁₁α were similar in the two strains, while the levels of G_sα (44 and 42 kDa forms) and the β-subunit were significantly reduced by ～20% in MHS membranes. The α-subunit of G_i3 was dramatically reduced by ～80% in MHS membranes. The affinities of β-adrenergic receptors for the antagonist, cyanophindolol, were similar in the two strains; however, the number of β-adrenoceptors was substantially reduced in MHS membranes. These findings may be of relevance to altered vascular reactivity and transmembrane ion distribution observed in the MHS.     

Prokineticin receptors interact unselectively with several G protein subtypes but bind selectively to β-arrestin 2

Prokineticin 1 (pk1) and prokineticin 2 (pk2) interact with two structurally related G-protein coupled receptors, prokineticin receptor 1 (PKR1) and prokineticin receptor 2 (PKR2). Cellular signalling studies show that the activated receptors can evoke Ca²⁺-mobilization, pertussis toxin-sensitive ERK phosphorylation, and intracellular cAMP accumulation, which suggests the partecipation of several G protein subtypes, such as G_q/11, G_i/o and G_s. However, direct interactions with these transduction proteins have not been studied yet. Here we measured by bioluminescence resonance energy transfer (BRET) the association of PKR1 and PKR2 with different heterotrimeric Gα proteins in response to pk1 and pk2 activation. Using host-cell lines carrying gene deletions of Gα_q/11 or Gα_s, and pertussis toxin treatment to abolish the receptor interactions with Gα_i/o, we determined that both receptors could couple with comparable efficiency to G_q/11 and G_i/o, but far less efficiently to G_s or other pertussis toxin-insensitive G proteins. We also used BRET methodology to assess the association of prokineticin receptors with β-arrestin isoforms. Fluorescent versions of the isoforms were transfected both in HEK293 cells and in double KO β-arrestin 1/2 mouse fibroblasts, to study receptor interaction with the reconstituted individual β-arrestins without background expression of the endogenous genes. Both receptors formed stable BRET-emitting complexes with β-arrestin 2 but not with β-arrestin 1, indicating strong selectivity for the former. In all the studied transducer interactions and in both receptors, pk2 was more potent than pk1 in promoting receptor binding to transduction proteins.     

Effect of activating and inactivating mutations of GS-and Gi2-alpha protein subunits on growth and differentiation of 3T3-L1 preadipocytes

Previous investigations have demonstrated that both G_s- and the G_i-family of GTP-binding proteins are implicated in differentiation of the 3T3-L1 preadipocyte. In order to further analyze the role of G_sα vs. G_i2α, which are both involved in adenylate cyclase modulation, we transfected undifferentiated 3T3-L1 cells with two sets of G-protein cDNA: the pZEM vector with either wild type, the activating (i.e., GTP-ase inhibiting) R201C-G_sα or the inactivating G226A(H21a)-G_sα point mutations, or the pZIPNeoSV(X) retroviral vector constructs containing the G_i2α wild type or the missense mutations R179E-G_i2α, Q205L-G_i2α, and G204A(H21a)-G_i2α. The activating [R201C]G_sα-mutant did not significantly affect the differentiation process, i.e., increase in the steady-state levels of G-protein subunits, gross appearance, or insulin-elicited deoxy-glucose uptake into 3T3-Ll adipocytes, despite a marked initial increase in hormone-elicited adenylate cyclase activity. The [H21a]G_sα-mutant, on the other hand, enhanced the degree of differentiation slightly, as evidenced by an augmented production of lipid vesicles and insulin-stimulated deoxy-glucose uptake. However, an expected increase in mRNA for hormone-sensitive lipase was not seen. Secondly, it appeared that both activating [R179E]G_i2α or [Q205L]G_i2α mutants reduced cell doubling time in non-confluent 3T3-L1 cell cultures, while [H21a]G_i2α slowed proliferation rate. Furthermore, it seemed that cell proliferation, as evidenced by thymidine incorporation, ceased at a much earlier stage prior to cell confluency when cultures were transfected with the [R179E]G_i2α or [Q205L]G_i2α mutants. Upon differentiation with insulin, dexamethasone, and iBuMeXan, the following cell characteristics emerged: the [R179E]G_i2α and [Q205L]G_i2α mutants consistently enhanced adenylate cyclase activation and cAMP accumulation stimulated by isoproterenol and corticotropin over controls. Deoxy-glucose uptake was also super-activated by the [R179E]G_i2α and [Q205L]G_i2α mutants. Finally, steady-state levels of hormone sensitive lipase mRNA were dramatically increased by [R179E]G_i2α and [Q205L]G_i2α over differentiated controls. The inactivating [H21a]G_i2α-mutant obliterated all signs of preadipocyte differentiation. It is concluded that G_i2 plays a positive and much more important role than G_s in 3T3-L1 preadipocyte differentiation. Cyclic AMP appears to play no role in this process. J. Cell. Biochem. 64:242–257. © 1997 Wiley-Liss, Inc.     

Identification and localization of G-proteins in the clonal adipocyte cell lines HGFu and Ob17

HGFu and Ob17 are cell lines derived from adipose tissue of lean (+/?) and ob/ob mice, respectively. Neither adenylyl cyclase activity nor G protein abundance and subcellular distribution have been assessed previously in these cells. Cyclase activity was low and resistant to catecholamine stimulation in both cell lines. However, the enzyme could be stimulated to high levels by forskolin and Mn²⁺. G_sα (largely the long isoform), G_iα2, and Gβ were the major G protein subunits identified. The levels of G protein mRNA expression were similar in both cell lines and, unlike actin expression, did not change as a result of differentiation. Immunoblotting and ADP-ribosylation of the G peptides corroborated these results. Assessment of the subcellular localization of the subunits by indirect epifluorescence and scanning confocal microscopy showed that each of the subunits had a characteristic subcellular pattern. G_sα showed vesicular cytoplasmic and nuclear staining; G_iα2 colocalized with actin stress fibers and disruption of these structures altered the distribution of G_iα2; β subunits showed some colocalization with the stress fibers as well as a cytoplasmic vesicular and nuclear pattern. As a result of differentiation, there was reorganization of the actin, together with the G_iα2 and β fibrous patterns. Both cell lines showed similar modifications. The induction of differentiation in these cells is therefore not associated with changes in adenylyl cyclase activity nor of the abundance of G-protein subunits, although reorganization of some of these subunits does accompany actin reorganization.     

A Gi2α antisense oligonucleotide differentiates morphine antinociception,constipation and acute dependence in mice

In the same mice in which the intracerebroventricular (i.c.v.) administration of antisense oligodeoxyribonucleotide (oligo) directed against the G_i2α (but not G_i1α, G_i3α or G_sα) G-protein subunits attenuated i.c.v. morphine-induced antinociception in the tail-flick test, none of the oligos altered naloxone-precipitated jumping (acute dependence). Likewise, none of the oligos significantly altered morphine-induced constipation. Hence, i.c.v. morphine-induced antinociception might be preferentially mediated via transduction pathway(s) different from constipation or acute dependence, offering novel opportunities for drug discovery.     

Elevated Stimulatory and Reduced Inhibitory G Protein α Subunits in Cerebellar Cortex of Patients with Dominantly Inherited Olivopontocerebellar Atrophy

Abstract: Although guanine nucleotide binding proteins (G proteins) are one of the critical components of signal transduction units for various membrane receptor-mediated responses, little information is available regarding their status in brain of patients with neurodegenerative illnesses. We measured the immunoreactivity of G protein subunits (G_sα, G_iα, G_oα, G_q/11α, and Gβ) in autopsied cerebellar and cerebral cortices of 10 end-stage patients with dominantly inherited olivopontocerebellar atrophy (OPCA) who all had severe loss of Purkinje cell neurons and climbing fiber afferents in cerebellar cortex. Compared with the controls, the long-form G_sα (52-kDa species) immunoreactivity was significantly elevated by 52% (p < 0.01) in the cerebellar cortex of the OPCA patients, whereas the G_i1α concentration was reduced by 35% (p < 0.02). No statistically significant differences were observed for G_oα, G_i2α, G_β1, G_β2, or G_q/11α in cerebellar cortex or for any G protein subunit in the two examined cerebral cortical subdivisions (frontal and occipital). The cerebellar G_sα elevation could represent a compensatory response (e.g., sprouting, reactive synaptogenesis) by the remaining cerebellar neurons (granule cells?) to neuronal damage but also might contribute to the degenerative process, as suggested by the ability of G_sα, in some experimental preparations, to promote calcium flux. Further studies will be required to determine the actual functional consequences of the G protein changes in OPCA and whether the elevated G_sα is specific to OPCA cerebellum, because of its unique cellular pattern of morphological damage, or is found in brain of patients with other progressive neurodegenerative disorders.     

α-Subunit G-protein antisense oligodeoxynucleotide effects on supraspinal (i.c.v.) α2-adrenoceptor antinociception in mice

An in vivo antisense strategy was used to examine the involvement of G-protein subunits in supraspinal (intracerebroventricular; i.c.v.) α₂-adrenoceptor-mediated antinociception. Mice that were injected with 33-mer antisense oligodeoxyribonucleotides (6 nmol) or vehicle were tested (tailflick) with an agonist (clonidine, guanfacine or BH-T 920) administered i.c.v. 18–24 h later. G_i3α antisense treatment attenuated BH-T 920 and clonidine-induced antinociception. G_i2α antisense produced differential effects on the three agonists. G_i1α and G_sα antisense treatment had no significant effect. Together with the previous demonstration that i.c.v. μ-opioid antinociception is mediated via G_i2α, the present results suggest that different receptors may mediate antinociception via different G-protein subunits and, hence, that specific subunits might offer novel targets for drug discovery.     

Molecular mechanisms of interaction of polycationic peptides with serpentine type receptors and heterotrimeric G-proteins in rat tissues

The key step in the hormonal signal transduction into cell is interaction of receptors with heterotrimeric G-proteins. We and other authors have shown that G-proteins may be activated as a result of their direct interaction with polycationic peptides. The goal of this work was to study molecular mechanisms of effect of hydrophobic peptide I, C-εAhx-WKK(C₁₀)-KKK(C₁₀)-KKKK(C₁₀)-YKK(C₁₀)-KK, and branched peptide II, [(GRGDSGRKKRRQRRRPPQ)₂-K-εAhx-C]₂ including the 48–60 fragment of the HIV-1 TAT-protein, on receptor and G-protein. These two peptides (10^?6?10^?4 M) produced a dose-dependent simulation of the GTP-binding activity of G-proteins in plasma membrane fractions of the brain striatum and cardiac muscle in rats. The effect of peptide I was more pronounced and decreased to a considerable degree in the presence of the C-terminal 385–394 peptide of the G-protein α_s-subunit that selectively disrupts interaction of receptors with G_s-protein. Peptide I reduced markedly affinity of serotonin (agonist) to the serotonin striatum receptors, whereas peptide II inhibited to the significant extent the binding of dihydroalprenolol (antagonist) to β-adrenergic receptors in cardiac muscle. Peptide I, unlike peptide II, decreased essentially the high affinity binding of β-agonist isoproterenol. The obtained data indicate the ability of polycationic peptides to activate G₁-proteins, to disturb their coupling with receptor, and to affect binding properties of the receptor. There are differences in molecular mechanisms of action of peptides with different structures on G-proteins and receptors.     

Determination of G-protein levels,ADP-ribosylation by cholera and pertussis toxins and the regulation of adenylyl cyclase activity in liver plasma membranes from lean and genetically diabetic (db/db)

Liver plasma membranes prepared from genetically diabetic (db/db) mice expressed levels of G_i α-2, G_i α-3 and G-protein β-subunits that were reduced by some 75, 63 and 73% compared with levels seen in membranes from lean animals. In contrast, there were no significant differences in the expression of the 42 and 45 kDa forms of G_s α-subunits. Pertussis toxin-catalysed ADP-ribosylation of membranes from lean animals identified a single 41 kDa band whose labelling was reduced by some 86% in membranes from diabetic animals. Cholera toxin-catalysed ADP-ribosylation identified two forms of G_s α-subunits whose labelling was about 4-fold greater in membranes from diabetic animals compared with those from lean animals. Maximal stimulations of adenylyl cyclase activity by forskolin (100 μM), GTP (100 μM), p[NH]ppG (100 μM), NaF (10 mM) and glucagon (10 μM) were similar in membranes from lean and diabetic animals, whereas stimulation by isoprenaline (100 μM) was lower by about 22%. Lower concentrations (EC₅₀-60 nM) of p[NH]ppG were needed to activate adenylyl cyclase in membranes from diabetic animals compared to those from lean animals (EC₅₀-158 nM). As well as causing activation, p[NH]ppG was capable of eliciting a pertussis toxin-sensitive inhibitory effect upon forskolin-stimulated adenylyl cyclase activity in membranes from both lean and diabetic animals. However, maximal inhibition of adenylyl cyclase activity in membranes from diabetic animals was reduced to around 60% of that found using membranes from lean animals. Pertussis toxin-treatment in vivo enhanced maximal stimulation of adenylyl cyclase by glucagon, isoprenaline and p[NH]ppG through a process suggested to be mediated by the abolition of functional G_i activity. The lower levels of expression of G-protein β-subunits, in membranes from diabetic compared with lean animals, is suggested to perturb the equilibria between holomeric and dissociated G-protein subunits. We suggest that this may explain both the enhanced sensitivity of adenylyl cyclase to stimulation by p[NH]ppG in membranes from diabetic animals and the altered ability of pertussis and cholera toxins to catalyse the ADP-ribosylation of G-proteins in membranes from these two animals.     

Pertussis toxin-sensitive G-proteins inhibit fibroblast growth factor-induced signaling in pancreatic acini

Signal transduction of fibroblast growth factor (FGF) receptors is known to involve tyrosine phosphorylation of several substrates, including Grb2, phospholipase C-γ, and phosphatidylinositol 3-kinase, whereas the role of G-proteins in FGF receptor signaling is controversial. In the present study we investigated the role of G-proteins in FGF receptor signaling in rat pancreatic acini. Immunological analysis revealed the presence of FGF receptor and phospholipase C-γ1 in rat pancreatic acini. Both basic fibroblast growth factor (FGF-2) and guanosine 5′-(γ-O-thio)triphosphate (GTPγS) caused an increase in inositol 1,4,5-trisphosphate (1,4,5-IP₃) production and amylase release. Combined stimulation of the acini with GTPγS and FGF-2 led to a decrease of these responses as compared to the effect of the single substances. When pancreatic acini were preincubated with FGF-2 (1 nM) or vehicle (water) ADP-ribosylation of the α-subunit of G_i-type G-proteins by pertussis toxin was reduced in membranes prepared from FGF-2 pretreated acini as compared to control acini, suggesting functional interaction of FGF receptors with G_i-proteins. Pretreatment of acini with pertussis toxin which inhibits G_i-type G-proteins abolished the inhibitory effect of GTPγS on FGF-induced 1,4,5-IP₃ production and amylase release, whereas the stimulatory effects of FGF-2 and GTPγS on these parameters remained unchanged. In conclusion, these results show communication of FGF receptors and G_i-type G-proteins and that G_i-type G-proteins exert an inhibitory influence on FGF-induced activation of phosphoinositide-specific phospholipase C in pancreatic acinar cells. © 1996 Wiley-Liss, Inc.     

Antagonist minigenes identify genes regulated by parathyroid hormone through G protein-selective and G protein co-regulated mechanisms in osteoblastic cells

Parathyroid hormone (PTH) is the major hormone regulating bone remodeling. Binding of PTH to the PTH1 receptor (PTH1R), a heterotrimeric G protein coupled receptor (GPCR), can potentially trigger multiple signal transduction pathways mediated through several different G proteins. In this study, we employed G protein antagonist minigenes inhibiting Gα_s, Gα_q or Gα₁₂ to selectively dissect out which of these G proteins were responsible for effects of PTH(1-34) in targeted signaling and osteogenesis arrays consisting of 159 genes. Among the 32 genes significantly regulated by 24 h PTH treatment in UMR-106 osteoblastic cells, 9 genes were exclusively regulated through G_s, 6 genes were solely mediated through G_q, and 3 genes were only controlled through G₁₂. Such findings support the concept that there is some absolute specificity in downstream responses initiated at the G protein level following binding of PTH to the PTH1R. On the other hand, 6 PTH-regulated genes were regulated by both G_s and G_q, 3 genes were regulated by both G_s and G₁₂, and 3 genes were controlled by G_s, G_q and G₁₂. These findings indicate potential overlapping or sequential interactions among different G protein-mediated pathways. In addition, two PTH-regulated genes were not regulated through any of the G proteins examined, suggesting that additional signaling mechanisms may be involved. Selectivity was largely maintained over a 2-48-hour time period. The minigene effects were mimicked by downstream inhibitors. The dissection of the differential effects of multiple G protein pathways on gene regulation provides a more complete understanding of PTH signaling in osteoblastic cells.     

Modulation of islet G-proteins, α-glucosidehydrolase inhibition and insulin release stimulated by various secretagogues

Guanine nucleotide-binding proteins (G-proteins) are known to act as important modulators of insulin release from the islets of Langerhans. We have recently found that the deoxynojirimycin-derivative emiglitate, a recognized inhibitor of intestinal -glucosidehydrolase activity, is a powerful inhibitor of glucose-induced insulin release. With the use of isolated mouse islets the present investigation was performed in a primary attempt to elucidate whether this inhibitory mechanism in some way was linked to the -cell G-protein system. Treatment of freshly isolated islets with pertussis toxin (PTX), which is known to inactivate the G_i-proteins, abolished the inhibitory effect of the ₂-adrenoceptor agonist clonidine on insulin release stimulated by the phosphodiesterase inhibitor IBMX in the presence of the protein kinase C activator TPA and even changed it into an increase. Emiglitate did not display any inhibitory action on insulin release induced by these secretagogues. Similarly, clonidine-induced inhibition of glucose stimulated insulin release was reversed by PTX. However, PTX did not influence the suppressive action of emiglitate on glucose-induced insulin secretion. In contrast, the adenylate cyclase activator forskolin totally abolished the inhibitory effect of emiglitate, but not that of the glucose analogue mannoheptulose, on glucose-induced insulin release. Moreover, the stimulatory effect of forskolin and cholera toxin (CTX) (activator of G_s-proteins) on the secretion of insulin was markedly enhanced in the presence of emiglitate. In conclusion, our results suggest that the inhibitory effect of emiglitate on glucose-induced insulin release is not directly related to the G_j-proteins, but most likely exerted solely through the selective suppression of lysosomal -glucosidehydrolase activity, a step in between the proximal and the distal G_i-proteins, in glucose-induced stimulus-secretion mechanisms. Our data also suggests that the inhibitory action of emiglitate on glucose stimulated insulin release can be compensated for by an increased sensitivity of the cyclic AMP-protein kinase A pathway. Hence, emiglitate might indirectly elicit an increased activity of the G_s-proteins to facilitate the secretory process.     

Functional dedifferentiation of parathyroid cells results both from a defective regulation and action of cytoplasmic Ca2+

Monolayer culture of bovine parathyroid cells for 24 hours resulted in a right-shift of the dose-effect relationships for Ca²⁺-inhibition of parathyroid hormone (PTH) release and the dependence of the cytoplasmic Ca²⁺ concentration (Ca²⁺) on extracellular Ca²⁺ as well as in a less suppressible hormone release. After 4 days of culture, hormone secretion was almost non-suppressible and Ca _i ²⁺ increased poorly in response to a rise in extracelluiar Ca²⁺. Ionomycin, a Ca²⁺ ionophore, raised Ca _i ²⁺ , but there was only a small inhibition of PTH release and the correlation between Ca _i ²⁺ and secretion was weak. A deteriorated Ca _i ²⁺ regulation and a decreased inhibitory action of cytoplasmic Ca²⁺ on PTH release were also found in ceils from human parathyroid adenomas. Functional dedifferentiation of the parathyroid cell thus results from both defective regulation and action of cytoplasmic Ca²⁺.     

Further evidence for direct pro-resorptive actions of FSH

We confirm that FSH stimulates osteoclast formation, function and survival to enhance bone resorption. It does so via the activation of a pertussis toxin-sensitive G_i-coupled FSH receptor that we and others have identified on murine and human osteoclast precursors and mature osteoclasts. FSH additionally enhances the production of several osteoclastogenic cytokines, importantly TNFα, likely within the bone marrow microenvironment, to augment its pro-resorptive action. FSH levels in humans rise before estrogen falls, and this hormonal change coincides with the most rapid rates of bone loss. On the basis of accumulating evidence, we reaffirm that FSH contributes to the rapid peri-menopausal and early post-menopausal bone loss, which might thus be amenable to FSH blockade.