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PubMed2001

Functional coupling of voltage-dependent L-type Ca2+ current to Ca2+-activated K+ current in pituitary GH3 cells.

Ca2+-activated K+ currents (I(K(Ca)) can contribute to action potential repolarization and after-hyperpolarization in GH3 cells. In this study, we examined how the activation of I(K(Ca) at the cellular level could be functionally coupled to Ca2+ influx through L-type Ca2+ channels. A 30-msec Ca2+ influx step to 0 mV was found to exhibit substantial contribution of Ca2+ influx through the activation of I(Ca,L) to the activation of I(K(Ca)). A bell-shaped relationship between the conditioning potentials and the integrated I(K(Ca)) was observed, suggesting that the magnitude of integrated I(Ca,L) correlates well with that of integrated I(K(Ca)) in the same cell. A linear relationship of integrated I(Ca,L) and integrated I(K(Ca)) was found with a coupling ratio of 69+/-7. The value of the coupling ratio was unaffected by the presence of Bay K 8644 or nimodipine, although these compounds could effectively affect the amplitudes of both I(K(Ca)) and I(Ca,L). However, tetrandrine could decrease the coupling ratio. Paxilline or intracellular Ca2+ buffer with EGTA decreased the coupling ratio, while apamin had no effect on it. Interestingly, phorbol 12-myristate 13-acetate also reduced the coupling ratio significantly, whereas thapsigargin increased this value. Thus, the present study indicates that the activation of I(K(Ca)) during brief Ca2+ influx, which is inhibited by paxilline, is coupled to Ca2+ influx primarily through the L-type channels. The selective modulation of I(K(Ca)) by second messengers or Ca2+ release from internal stores may affect the coupling efficiency and hence cellular excitability.

Alkaloids↗

PubMed2002

Couple communication and female problem drinking: a behavioral observation study.

Couples with alcohol and relationship problems often report poor communication, yet little is known about the communication of maritally distressed couples in which the woman abuses alcohol (MDWA couples). Compared with maritally distressed couples without alcohol problems (MDNA) and couples with neither problem (NDNA), MDWA couples showed a distinctive pattern of negative communication. Similar to MDNA men, MDWA men spoke negatively to their partners but listened positively to their partners much like NDNA men. MDWA women listened negatively, much as MDNA women did, but spoke positively, like NDNA women did. The interactions of MDWA couples can be characterized as a male-demand-female-withdraw pattern, which is a gender reversal of the female-demand-male-withdraw pattern often observed in MDNA couples.

Adult↗

PubMed2004

Hierarchy of carcinoma cell responses to apigenin: gap junctional coupling versus proliferation.

We discriminated the role of gap junctional communication and phenotypic constitution of tumour cells in determining their responsiveness to apigenin. Effects of apigenin on intercellular communication and proliferation of two lines of carcinoma cells, uncoupled HeLa cells and their coupled Cx43-transfected counterparts, were analysed and compared with the responses of highly coupled BICR/M1Rk cells. Dye transfer analyses demonstrated that apigenin decreases the degree of coupling in Cx43-coupled populations of HeLa cells but does not affect BICR/M1Rk cells. Similarly, no communication enhancement was observed in originally uncoupled HeLa cell populations. A G2-specific growth arrest paralleled by the induction of apoptosis was observed which was more pronounced and correlated with higher number of apoptotic events in coupled HeLa Cx43 transfectants than in parental cell line. On the other hand, apoptosis was not observed in highly coupled BICR/M1Rk cells, instead, these cells were only transiently blocked in G2 which might be a result of their ability to metabolise apigenin. These data demonstrate a hierarchy of systems determining cellular sensitivity to apigenin. Gap junctional coupling does not influence the quality of cell cycle-related responses to apigenin but modulates their magnitude. This modulating effect of gap junctional coupling depends, however, on a cellular context determined by specific cell phenotype and can be overcome by tissue-specific compensatory mechanisms.

Animals↗

PubMedDate not supplied

The effect of treatment on pregnancy among couples with unexplained infertility.

Our objective was to determine the effect of treatment on the likelihood of pregnancy among couples with unexplained infertility. We used a nonrandomized, prospective, multicentered cohort analytic study, with mean follow-up time of 14.5 months (range, 0.5-46 months). The subjects were 470 couples who attended infertility clinics affiliated with medical schools in Canada, in whom no abnormality was found after investigation. They were drawn from a total of 2,106 couples registered from April 1, 1984 to March 31, 1987. Of these, 130 couples were selected for treatment at the discretion of the care givers; 340 couples were not treated. Selection for treatment resulted in imbalance between the groups: the treated couples had a longer mean duration of infertility (48 vs. 36 months), and were more likely to have had a laparoscopy as part of the investigation (72% vs. 48%). No specific protocol of treatment was used. Treatments most commonly used were clomiphene (87); gonadotropins (31); intrauterine insemination (20); IVF or GIFT (16); bromocriptine (12); 43 couples had two treatments, and 11 had three treatments. The only important determinants of treatment (logistic regression) were time under observation and laparoscopy status. Duration of infertility was only a minor determinant of treatment. Crude, unadjusted pregnancy rates were 25% for the treated group and 34% for the untreated group. The early occurrence of pregnancy in the untreated couples accounted for much of this difference. After adjustment for baseline differences between the groups and times to and under treatment with proportional hazards analysis, the cumulative probability of pregnancy is 2.0 (95% CI 1.3 to 3.1) times as high with treatment.(ABSTRACT TRUNCATED AT 250 WORDS)

Adult↗

PubMed1986

Enhancement of antigenic potency in vitro and immunogenicity in vivo by coupling the antigen to anti-immunoglobulin.

We have examined the effect of targeting an antigen to the immune system, by covalently coupling it to anti-immunoglobulin (Ig), on its efficacy for T cell stimulation in vitro and its immunogenicity for antibody production in vivo. In vitro, we compared the potency (for stimulation of a ferritin-specific T cell line) of free ferritin, ferritin coupled to goat antimouse IgM (heavy (H) chain specific), ferritin coupled to anti-IgG (H and light (L) chain specific), or ferritin coupled to anti-IgA (H chain specific), as well as a mixture of free ferritin plus goat anti-IgG. The ferritin coupled to anti-IgM or to anti-IgG (H + L), which could bind to surface Ig of B cells, stimulated T cell proliferation at concentrations of ferritin at least 10-fold lower than those required for the other forms of the antigen over the entire time course of the response, with 1000 rad-irradiated spleen cells as presenting cells. Because the goat antibodies were all of the same IgG isotype and coupling ratio, the failure of goat anti-IgA to enhance potency served as a control to exclude Fc receptor binding as the mechanism. The effect was not due to the nonspecific activation of B cells to become more efficient antigen-presenting cells, because mixtures of ferritin plus anti-IgG (H + L) had no effect, and the anti-IgG coupled to ferritin did not enhance presentation of myoglobin to a myoglobin-specific T cell line. The enhanced presentation of ferritin conjugated to goat anti-IgG (H + L) or to anti-IgM was sensitive to radiation doses greater than 2000 R, and was effective at less than one-tenth the number of spleen cells, consistent with the predominance of B cells as antigen-presenting cells for this form of the antigen rather than macrophages and dendritic cells only. When B cells and accessory cells were purified from T-depleted spleen cells, only the B cell preparation but not the accessory cell population manifested enhanced presentation of ferritin coupled to anti-IgG compared with free ferritin, and it was radiosensitive. Finally, allogeneic B cells could not mediate the enhancement in the presence of syngeneic splenic accessory cells (SAC); therefore, the enhancement was not due to shedding of immune complexes from B cells and subsequent presentation by SAC. We conclude that targeting the antigen to B cells as presenting cells greatly enhances its efficacy in vitro.(ABSTRACT TRUNCATED AT 400 WORDS)

Animals↗

PubMed1987

Managing both members of a two-pharmacist couple: the employer's perspective.

Employer considerations in hiring and managing the two-pharmacist couple are discussed. In recruiting one member of a two-pharmacist couple, an employer may need to consider hiring the other spouse, especially if the employment requires relocation and if the institution is the only practice site in the area. Managers should be aware of potential difficulties associated with this practice, such as the potential for formal charges if illegal discrimination is perceived as a motive for selecting a candidate. If one spouse is not acceptable, the employer could provide assistance in locating another position. In some organizations, family members cannot supervise, or even work with, one another. Employers should review policies concerning organizational working relationships with two-pharmacist couples. In terms of scheduling, two-pharmacist couples may have work-schedule preferences that are difficult to accommodate. Although organizations have traditionally designed compensation packages for individuals, some nonsalary benefits may be handled more efficiently on a family basis. Problems can arise when individuals in two-pharmacist couples are promoted at different times and frequencies. This possibility should be discussed with both employees at the time of hiring and reviewed as promotions occur. The resignation or dismissal of one spouse may lead to the departure of the other. If this type of staff loss occurs frequently, other staff may perceive the hiring of two-pharmacist couples as a poor management practice. When hiring and managing both spouses in a dual-career couple, managers can minimize potential problems by maintaining good communication with the couple and other staff.

Employment↗

PubMed1986

Demographic and personality characteristics of couples undergoing in vitro fertilisation.

Two hundred and fifty-eight (258) couples requesting in vitro fertilisation (IVF) were interviewed by a social worker prior to commencing treatment. At the time of admission to hospital 147 women and 134 men completed a questionnaire which incorporated the Eysenck Personality Inventory and the Spielberger State-Trait Anxiety Inventory. Forty-three women conceived and 26 couples (14.2%) required extra counselling or support after their involvement in the programme. Couples seeking IVF did not differ significantly from the rest of the population with respect to occupation, education, alcohol, cigarette and medication use. The personality characteristics of couples seeking IVF did not differ significantly from a local comparison group. Personality and psychosocial characteristics did not identify those couples who were more likely to become pregnant by IVF. However, couples who required extra counselling were more likely to have required similar help in the past. Women requiring counselling also scored significantly higher on the Eysenck neuroticism scale and the Spielberger State-Trait anxiety scales compared to those women who did not require extra counselling or support. The male partners scored significantly lower on the Eysenck scale of extroversion compared to male partners of couples who did not require support or counselling. We conclude that these inventories may be of value in identifying certain couples who require extra care during attempted conception by IVF.

Adult↗

PubMed1993

Hydrophobic amino acid in the i2 loop plays a key role in receptor-G protein coupling.

Signal transduction of the heptahelical G protein-coupled receptors (GPCRs) involves multiple receptor domains, but a universal consensus domain for coupling has not yet been defined. Alanine mutagenesis scanning was performed on the intracellular loops and the COOH tail of the human muscarinic cholinergic receptor (Hm1) to identify coupling domains. Stimulation of phosphatidylinositol (PI) turnover was determined after transfection of the alanine mutants into U293 human embryonic kidney cells. Alanine substitutions in four regions (loops i1, i2, and NH2 and COOH junctions of i3) impaired coupling efficiency by approximately 50% or more, but the strongest reduction (> 80%) resulted from alanine replacement of a single amino acid, leucine 131. This residue is located in the middle of the second intracellular loop (i2), within the highly conserved GPCR motif (DRYXXV(I)XXPL). The position equivalent to Leu-131 in Hm1 contains a bulky hydrophobic amino acid (L, I, V, M, or F) in nearly all cloned GPCRs. Substitution of Leu-131 with polar amino acids (aspartate and asparagine) also resulted in strongly defective coupling, whereas phenylalanine (found in the equivalent position in the beta 2 adrenoceptor) can replace leucine without losing PI coupling ability of Hm1. Alanine substitution of the corresponding amino acid in the Hm3 receptor (L174A) also inhibited agonist-stimulated PI turnover, while replacing Phe-139 with alanine in the beta 2 adrenoceptor suppressed stimulation of adenylyl cyclase. We propose that a bulky hydrophobic amino acid in the middle of the i2 loop serves as a general site relevant to G protein coupling, whereas coupling selectivity is governed by other receptor domains.

Amino Acid Sequence↗

PubMed1993

[Membrane receptors and coupling proteins in adenohypophyseal cells].

Dopamine receptors of D2 type present on lactotroph cells are coupled to a large series of transduction mechanisms. Beside their negative coupling with adenylate cyclase, they are also coupled with potassium and calcium channels, leading to a decreased intracellular calcium concentration. In addition, D2 dopamine receptors also modulate phospholipase activities. Dopamine inhibits inositol phosphate production, through two distinct mechanisms. One of them could represent a direct negative coupling with phospholipase C. All these transduction mechanisms of the D2 dopamine receptors implicate G proteins sensitive to pertussis toxin. In contrast, these receptors are negatively coupled to phospholipase A2 through G proteins insensitive to this toxin. Both isoforms of the D2 dopamine receptor, generated by alternate splicing of a single gene are present in lactotroph cells. After transfection in CH4C1 cells the two isoforms are coupled with adenylate cyclase while only the shortest isoform appears negatively coupled to phospholipase C. Functional D2 dopamine receptors are present in human prolactinomas. Resistance to bromocriptine therapy is associated with a decrease density of these receptors in the tumor. In addition, the ratio of the two receptor isoforms (measured by PCR) is different in responsive and resistant tumors. Furthermore, the activity of Gi/Go proteins coupled to adenylate cyclase appears also affected in resistant tumors. Resistance to bromocriptine therapy appears thus to involve multiple changes at the different levels of the multiple mechanisms of action of dopamine on lactotroph cells.

Animals↗

PubMed1993

Dopaminergic modulation of dye coupling between neurons in the core and shell regions of the nucleus accumbens.

The presence of dye coupling and electrotonic coupling within the nucleus accumbens was examined using intracellular recording and staining in rat brain slices. In 24% of the cells examined, injection of single accumbens neurons with the dye Lucifer yellow resulted in the complete labeling of more than one cell. To examine whether this coupling is regulated by dopamine (DA), the extent of dye coupling was assessed in the presence of dopaminergic agonists and antagonists. In the core region of the accumbens, D1 receptor stimulation induced a decrease in the incidence of dye coupling; this effect could be blocked by the D1 antagonists SCH 23,390 and SCH 39,166, by the D2 antagonist sulpiride, or by depletion of DA by reserpine and alpha-methyl-p-tyrosine coadministration, but not by the atypical antipsychotic drug clozapine. In contrast, in the posterior part of the shell region, D2 receptor stimulation increased the incidence of coupling independent of the state of D1 receptor activation. Fast spikelets resembling coupling potentials were found to occur only in neurons that also showed dye coupling, and could be attenuated in core neurons by D1 receptor stimulation. These findings suggest that DA, in addition to its actions on the membrane of single neurons, may also affect neuronal integration on a network level by regulating the transfer of information between adjacent neurons within the nucleus accumbens.

Action Potentials↗

PubMed1993

Mutations of the alpha 2A-adrenergic receptor that eliminate detectable palmitoylation do not perturb receptor-G-protein coupling.

The alpha 2A-adrenergic receptor (alpha 2AAR) is coupled to a variety of effectors via pertussis toxin-sensitive GTP-binding proteins. Like most members of the G-protein-coupled receptor superfamily, the primary structure of the alpha 2AAR possesses a putative consensus sequence for palmitoylation in the COOH terminus at Cys-442. This study demonstrates that the alpha 2AAR incorporates [3H] palmitic acid in metabolic labeling studies and that mutation of Cys-442 to Ala or Ser eliminates detectable 3H-palmitoylation. However, mutation of Cys-442 does not alter adrenergic ligand specificity or allosteric modulation by amphipathic agents, such as amiloride analogs. Since reports in the literature suggest that a homologous mutation in the beta 2-adrenergic receptor attenuates coupling to Gs (O'Dowd, B. F., Hnatowich, M., Caron, M. G., Lefkowitz, R. J., and Bouvier, M. (1989) J. Biol. Chem. 264, 7564-7569) whereas chemical removal of palmitate from bovine rhodopsin enhances coupling to Gt (Morrison, D. F., O'Brien, P. J., and Pepperberg, D. R. (1991) J. Biol. Chem. 266, 20118-20123), we examined if mutation of Cys-442 and parallel loss of detectable palmitoylation alter alpha 2AAR coupling to G-proteins. Several independent cell lines of Madin-Darby canine kidney II cells expressing wild-type (Cys-442) or mutant (Ala-442 and Ser-442) alpha 2AARs were established. Metabolic labeling of Madin-Darby canine kidney cells expressing wild-type (Cys-442) or mutant (Ala-442) alpha 2AARs with [3H]palmitic acid indicated that only wild-type Cys-442-containing receptors incorporated [3H]palmitate, monitored following isolation of the alpha 2AAR detergent extracts using yohimbine-agarose chromatography. Receptor-G-protein coupling was assessed by evaluating sensitivity of receptor-agonist interactions to guanine nucleotides in competition for [3H]yohimbine antagonist binding, guanyl-5'-yl imidotrisphosphate sensitivity of pertussis toxin-sensitive p-[125I]iodoclonidine agonist binding, and agonist-stimulated guanosine 5'-O-(thiotriphosphate) binding. Using all three approaches, no detectable change in alpha 2AAR-G-protein coupling was apparent, in contrast to apparent opposite effects on the beta 2-adrenergic receptor-Gs and rhodopsin-Gt coupling reported previously by others. One interpretation is that this conserved cysteine may play differing roles at different receptor-G-protein interfaces. Alternatively, this shared structural motif may play a role in not yet investigated pathways, such as receptor expression, turnover, and localization.

Acylation↗

PubMed1996

Gi-mediated stimulation of type II adenylyl cyclase is augmented by Gq-coupled receptor activation and phorbol ester treatment.

Synergism between Gs- and Gi- or Gq-dependent signaling pathways has been demonstrated in the stimulation of type II adenylyl cyclase (AC-II). Provision of activated alpha s is known to allow numerous Gi-coupled receptors to stimulate AC-II and to potentiate the responses to Gq-coupled receptors. To explore possible interactions between Gi- and Gq-coupled receptors that are independent of alpha s, the activity of AC-II was determined after the activation of Gi- and Gq-regulated pathways. Human embryonic kidney 293 cells were transiently cotransfected with cDNAs encoding AC-II and various G-protein-coupled receptors. Agonist-bound Gi-coupled receptors (including the formyl peptide, dopamine-D2, and delta-opioid receptors) stimulated AC-II activity in the absence of activated alpha s, provided that the cells were treated with 100 nM phorbol 12-myristate 13-acetate. Activation of protein kinase C (PKC) thus appears to relieve the requirement for the presence of activated alpha s. Stimulation of PKC via Gq-coupled receptors also allowed Gi-coupled receptors to activate AC-II. Coexpression of the m1 muscarinic receptor with the dopamine-D2 receptor permitted dopamine to stimulate AC-II in the presence of carbachol. The phorbol ester-permissive and alpha s-independent stimulation was mediated by G-protein beta gamma subunits because it was blocked by the beta gamma scavengers alpha t and beta-adrenergic receptor kinase. These results show that AC-II can efficiently integrate signals generated by Gq- and Gi-coupled receptors via a mechanism that is independent of Gs.

Adenylyl Cyclases↗

PubMed1998

Functional coupling of creatine kinases in muscles: species and tissue specificity.

Creatine kinase (CK) isoenzymes are present in all vertebrates. An important property of the creatine kinase system is that its total activity, its isoform distribution, and the concentration of guanidino substrates are highly variable among species and tissues. In the highly organized structure of adult muscles, it has been shown that specific CK isoenzymes are bound to intracellular compartments, and are functionally coupled to enzymes and transport systems involved in energy production and utilization. It is however, not established whether functional coupling and intracellular compartmentation are present in all vertebrates. Furthermore, these characteristics seem to be different among different muscle types within a given species. This study will review some of these aspects. It has been observed that: (1) In heart ventricle, CK compartmentation and coupling characterize adult mammalian cells. It is almost absent in frogs, and is weakly present in birds. (2) Efficient coupling of MM-CK to myosin ATPase is seen in adult mammalian striated muscles but not in frog and bird heart where B-CK is expressed instead of M-CK. Thus, the functional efficacy of bound MM-CK to regulate adenine nucleotide turnover within the myofibrillar compartment seems to be specific for muscles expressing M-CK as an integral part of the sarcomere. (3) Mi-CK expression and/or functional coupling are highly tissue and species specific; moreover, they are subject to short term and long term adaptations, and are present late in development. The mitochondrial form of CK (mi-CK) can function in two modes depending on the tissue: (i) in an < > and (ii) in an < >. The mode of action of mi-CK seems to be related to its precise localization within the mitochondrial intermembrane space, whereas its amount might control the quantitative aspects of the coupling. Mi-CK is highly plastic, making it a strong candidate for fine regulation of excitation-contraction coupling in muscles and for energy transfer in cells with large and fluctuating energy demands in general. (4) Although CK isoforms show a binding specificity, the presence of a given isoform within a tissue or a species only, does not predict its functional role. For example, M-CK is expressed before it is functionally compartmentalized within myofibrils during development. Similarly, the presence of ubiquitous or sarcomeric mi-CK isoforms, is not an index of functional coupling of mi-CK to oxidative phosphorylation. (5) Amongst species or muscles, it appears that a large buffering action of the CK system is associated with rapid contraction and high glycolytic activity. On the other hand, an oxidative metabolism is associated with isoform diversity, increased compartmentation, a subsequent low buffering action and efficient phosphotransfer between mitochondria and energy utilization sites. It can be concluded that, in addition to a high variation of total activity and isoform expression, the role of the CK system also critically depends on its intracellular organization and interaction with energy producing and utilizing pathways. This compartmentation will determine the high cellular efficiency and fine specialization of highly organized and differentiated muscle cells.

Adenine Nucleotides↗

PubMed2005

Treating infidelity in same-sex couples.

Psychotherapy with same-sex couples does not differ markedly from standard couple therapies; this is also true for treating couples facing infidelity. However, same-sex couples often design their relationships differently, without tradition and formal marital contracts to prescribe behavior. Based on clinical experience and the empirical research, this article addresses the differing norms involved in affirmatively treating infidelity in gay and lesbian couples within the framework of integrative behavioral couple therapy (IBCT). Two cases illustrate the process and outcome of IBCT with same-sex couples.

Adult↗

PubMed1998

Human dopamine D3 and D2L receptors couple to inward rectifier potassium channels in mammalian cell lines.

The molecular mechanisms coupling the D3 dopamine receptor to downstream effectors have neither been well defined nor well characterized. Here we examine the coupling of the human D3 receptor to G-protein coupled inward rectifier potassium channels (GIRKs) in mammalian cells. Human D3 receptors couple strongly to homomeric human GIRK2 channels coexpressed in Chinese hamster ovary (CHO) cells, with a coupling efficiency comparable to that of D2L receptors. The coupling between D3 receptors and native GIRK channels was examined in an AtT-20 mouse pituitary cell line stably expressing the human D3 receptor. AtT-20 cells endogenously express somatostatin and muscarinic receptors coupled to GIRK channels. RT-PCR and Western blot analyses revealed that AtT-20 cells natively express Kir3.1 and Kir3.2 channel isoforms, but not D2 or D3 dopamine receptors. In D3 receptor expressing AtT-20 cells, application of the D2/D3 receptor agonist, quinpirole, induces pertussis toxin-sensitive inward rectifying K+ currents that are blocked by barium. Activation of D3 receptors leads to both homologous desensitization of this receptor and an unusual unidirectional heterologous desensitization of somatostatin receptors. AtT-20 cells may be a good model to examine the functional role of D3 dopamine receptors in regulating neurotransmitter secretion.

Adenosine Triphosphate↗

PubMed2005

Ultra-low-dose naloxone suppresses opioid tolerance, dependence and associated changes in mu opioid receptor-G protein coupling and Gbetagamma signaling.

Opiates produce analgesia by activating mu opioid receptor-linked inhibitory G protein signaling cascades and related ion channel interactions that suppress cellular activities by hyperpolarization. After chronic opiate exposure, an excitatory effect emerges contributing to analgesic tolerance and opioid-induced hyperalgesia. Ultra-low-dose opioid antagonist co-treatment blocks the excitatory effects of opiates in vitro, as well as opioid analgesic tolerance and dependence, as was demonstrated here with ultra-low-dose naloxone combined with morphine. While the molecular mechanism for the excitatory effects of opiates is unclear, a switch in the G protein coupling profile of the mu opioid receptor and adenylyl cyclase activation by Gbetagamma have both been suggested. Using CNS regions from rats chronically treated with vehicle, morphine, morphine+ultra-low-dose naloxone or ultra-low-dose naloxone alone, we examined whether altered mu opioid receptor coupling to G proteins or adenylyl cyclase activation by Gbetagamma occurs after chronic opioid treatment. In morphine-naïve rats, mu opioid receptors coupled to Go in striatum and to both Gi and Go in periaqueductal gray and spinal cord. Although chronic morphine decreased Gi/o coupling by mu opioid receptors, a pronounced coupling to Gs emerged coincident with a Gbetagamma interaction with adenylyl cyclase types II and IV. Co-treatment with ultra-low-dose naloxone attenuated both the chronic morphine-induced Gs coupling and the Gbetagamma signaling to adenylyl cyclase, while increasing Gi/o coupling toward or beyond vehicle control levels. These findings provide a molecular mechanism underpinning opioid tolerance and dependence and their attenuation by ultra-low-dose opioid antagonists.

Adenylyl Cyclases↗

PubMed2003

Coupling to Gs and G(q/11) of histamine H2 receptors heterologously expressed in adult rat atrial myocytes.

The predominant histamine receptor subtype in the supraventricular and ventricular tissue of various mammalian species is the H2 receptor (H2-R) subtype, which is known to couple to stimulatory G proteins (Gs), i.e. the major effects of this autacoid are an increase in sinus rate and in force of contraction. To investigate histamine effects in H2-R-transfected rat atrial myocytes, endogenous GIRK currents and L-type Ca2+ currents were used as functional assays. In H2-R-transfected myocytes, exposure to His resulted in a reversible augmentation of L-type Ca2+ currents, consistent with the established coupling of this receptor to the Gs-cAMP-PKA signalling pathway. Mammalian K+ channels composed of GIRK (Kir3.x) subunits are directly controlled by interaction with betagamma subunits released from G proteins, which couple to seven-helix receptors. In mock-transfected atrial cardiomyocytes, activation of muscarinic K+ channels (IK(ACh)) was limited to Gi-coupled receptors (M2R, A1R). In H2-R-overexpressing cells, histamine activated IK(ACh) via Gs-derived betagamma subunits since the histamine-induced current was insensitive to pertussis toxin. These data indicate that overexpression of Gs-coupled H2-R results in a loss of target specificity due to an increased agonist-induced release of Gs-derived betagamma subunits. When IK(ACh) was maximally activated by GTP-gamma-S, histamine induced an irreversible inhibition of the inward current in a fraction of H2-R-transfected cells. This inhibition is supposed to be mediated via a G(q/11)-PLC-mediated depletion of PIP2, suggesting a partial coupling of overexpressed H2-R to G(q/11). Dual coupling of H2-Rs to Gs and Gq is demonstrated for the first time in cardiac myocytes. It represents a novel mechanism to augment positive inotropic effects by activating two different signalling pathways via one type of histamine receptor. Activation of the Gs-cAMP-PKA pathway promotes Ca2+ influx through phosphorylation of L-type Ca2+ channels. Simultaneous activation of Gq-signalling pathways might result in phosphoinositide turnover and Ca2+ release from intracellular stores, thereby augmenting H2-induced increases in [Ca2+]i.

Animals↗

PubMed1998

The subcellular and cellular distribution of G protein-coupled receptor kinase 2 in rat brain.

G protein-coupled receptor kinase 2 has been found to phosphorylate and thus regulate the activity of several G protein-coupled receptors implicated in neuronal signalling pathways. Although this kinase was initially described as a soluble protein, our laboratory has recently found that a significant amount of G protein-coupled receptor kinase 2 is associated with microsomal membranes in liver and different types of cultured cells. In the present report we show that high G protein-coupled receptor kinase 2 specific activity and protein levels are present in microsomal fractions of rat brain homogenates. On the other hand, immunochemical detection using a new antibody raised against the N-terminus of the kinase revealed a specific and widely distributed staining in different areas of the central nervous system, and the association of G protein-coupled receptor kinase 2 with intracellular structures in nervous cells. Our results further suggest that this receptor kinase may be involved in the modulation of G protein-coupled receptor-mediated neurotransmission and that association with microsomal membranes may play a role in G protein-coupled receptor kinase 2 functions in the brain.

Animals↗