The impact of patients as trainers on registered nurses' patient engagement in primary care clinics: a qualitative study.

BACKGROUND: In Canada, primary care is usually the front door to health care for people with health issues. Among these primary care services are primary care clinics (PCC), where the competencies of registered nurses (RNs) are needed. However, nursing practice in PCCs is variable and sometimes suboptimal from one PCC to another. In 2019, the Quebec Ministry of Health and Social Services deployed a practical guide for RNs practicing in PCCs. This guide was intended to support best professional and interprofessional practices and enhance the quality of services offered according to a physical-social vision of care, interprofessional collaboration and partnership with the patient. The Formation de formateurs en première ligne (F2PL) project team developed a train-the-trainer educational intervention to support RNs in assimilating the content of this guide. This educational intervention is uncommon because it includes patients as trainers (PTs). PTs developed and provided andragogic content about patient's experience to enhance patient engagement. OBJECTIVE: To describe the impacts of the educational intervention provided by the PTs in nurses' patient engagement practices in PCCs. METHODS: A descriptive qualitative approach was used to describe in-depth changes in RNs' practices. Individual interviews were conducted with 10 RNs and 3 PTs to explore the changes in RNs' practice and the barriers and facilitators to adopting this new practice. An inductive and deductive thematic analysis was carried out according to a conceptual model of patient engagement (the Montreal Model), and emerging themes were condensed into propositions. To ensure credibility, a peer review was conducted with the F2PL team, which includes a patient co-leader. RESULTS: The educational intervention provided by PTs has impacted RNs' practice in 3 ways: awareness or reminding of general principles, updating commitment to already known principles and enhancing the development of new professional skills. CONCLUSIONS: PTs could effectively support the RNs' motivation to use patient engagement practices in primary care.

Neuropsychological Sequelae of Coronary Heart Disease in Women: A Systematic Review.

Heart disease, such as coronary heart disease (CHD), is the leading cause of death among aging women. However, over the past years, the mortality rate has declined, resulting in an increased number of CHD survivors. In this context, research has uncovered relationships between cardiovascular disease (CVD) and the development of neurodegenerative diseases, suggesting that CHD can act as a precursor. Despite heart disease affecting both sexes, CVD research has significantly neglected women. Therefore, we conducted the first systematic review of neuropsychological sequelae of CHD in women to gain a clear portrait of the current knowledge of the association of CHD on women's neuropsychological status. We found that studies continue to include an insufficient number of women in their research. Our work also uncovered that there is variability in the definition of CHD by researchers (i.e., operationalization of the variable), which could explain inconsistencies across studies. Overall, we found evidence that supports the heart-brain disease hypothesis. To conclude, we provide several guidelines for future research involving the impact of CHD in women.

Spatial and functional relationship between poly(ADP-ribose) polymerase-1 and poly(ADP-ribose) glycohydrolase in the brain.

Poly(ADP-ribose) polymerases (PARPs) are members of a family of enzymes that utilize nicotinamide adenine dinucleotide (NAD(+)) as substrate to form large ADP-ribose polymers (PAR) in the nucleus. PAR has a very short half-life due to its rapid degradation by poly(ADP-ribose) glycohydrolase (PARG). PARP-1 mediates acute neuronal cell death induced by a variety of insults including cerebral ischemia, 1-methyl-4-phenyl-1,2,3,6-tetrahydropyridine-induced Parkinsonism, and CNS trauma. While PARP-1 is localized to the nucleus, PARG resides in both the nucleus and cytoplasm. Surprisingly, there appears to be only one gene encoding PARG activity, which has been characterized in vitro to generate different splice variants, in contrast to the growing family of PARPs. Little is known regarding the spatial and functional relationships of PARG and PARP-1. Here we evaluate PARG expression in the brain and its cellular and subcellular distribution in relation to PARP-1. Anti-PARG (alpha-PARG) antibodies raised in rabbits using a purified 30 kDa C-terminal fragment of murine PARG recognize a single band at 111 kDa in the brain. Western blot analysis also shows that PARG and PARP-1 are evenly distributed throughout the brain. Immunohistochemical studies using alpha-PARG antibodies reveal punctate cytosolic staining, whereas anti-PARP-1 (alpha-PARP-1) antibodies demonstrate nuclear staining. PARG is enriched in the mitochondrial fraction together with manganese superoxide dismutase (MnSOD) and cytochrome C (Cyt C) following whole brain subcellular fractionation and Western blot analysis. Confocal microscopy confirms the co-localization of PARG and Cyt C. Finally, PARG translocation to the nucleus is triggered by NMDA-induced PARP-1 activation. Therefore, the subcellular segregation of PARG in the mitochondria and PARP-1 in the nucleus suggests that PARG translocation is necessary for their functional interaction. This translocation is PARP-1 dependent, further demonstrating a functional interaction of PARP-1 and PARG in the brain.

NMDA but not non-NMDA excitotoxicity is mediated by Poly(ADP-ribose) polymerase.

Poly(ADP-ribose) polymerase (PARP-1), a nuclear enzyme that facilitates DNA repair, may be instrumental in acute neuronal cell death in a variety of insults including, cerebral ischemia, 1-methyl-4-phenyl-1,2,3,6-tetrahydropyridine-induced parkinsonism, and CNS trauma. Excitotoxicity is thought to underlie these and other toxic models of neuronal death. Different glutamate agonists may trigger different downstream pathways toward neurotoxicity. We examine the role of PARP-1 in NMDA- and non-NMDA-mediated excitotoxicity. NMDA and non-NMDA agonists were stereotactically delivered into the striatum of mice lacking PARP-1 and control mice in acute (48 hr) and chronic (3 week) toxicity paradigms. Mice lacking PARP-1 are highly resistant to the excitoxicity induced by NMDA but are as equally susceptible to AMPA excitotoxicity as wild-type mice. Restoring PARP-1 protein in mice lacking PARP-1 by viral transfection restored susceptibility to NMDA, supporting the requirement of PARP-1 in NMDA neurotoxicity. Furthermore, Western blot analyses demonstrate that PARP-1 is activated after NMDA delivery but not after AMPA administration. Consistent with the theory that nitric oxide (NO) and peroxynitrite are prominent in NMDA-induced neurotoxicity, PARP-1 was not activated in mice lacking the gene for neuronal NO synthase after NMDA administration. These results suggest a selective role of PARP-1 in glutamate excitoxicity, and strategies of inhibiting PARP-1 in NMDA-mediated neurotoxicity may offer substantial acute and chronic neuroprotection.

Two Ca2+ transport systems are distinguished on the basis of their Mg2+ dependency in a post-nuclear particulate fraction of bovine adrenal cortex.

Inositol 1,4,5-trisphosphate (InsP3) is a second messenger responsible for Ca2+ release from an internal store whose nature and location remains undefined. To get more information on this intracellular Ca2+ store, a post-nuclear particulate fraction was prepared from bovine adrenal cortex and its Ca2+ uptake and release activities were monitored with the fluorescent indicator Fura-2. In the presence of Mg2+ (2 mM), the particulate preparation showed high ATP-dependent Ca2+ sequestering activity and decreased the ambient Ca2+ concentration to about 150 nM. In the absence of Mg2+, Ca2+ was still sequestered but less efficiently, reaching a level around 170 nM. In the presence of Mg2+, the Ca2+ released by a maximal dose of InsP3 (2 microM) was completely resequestered whereas in the absence of Mg2+, no resequestration occurred even after complete degradation of InsP3. The use of selective agents such as oligomycin, saponin, ionomycin and biliary salts indicated that Ca2+ was stored in three different pools which are distinct from the mitochondria and from inside-out membrane vesicles. Our data also indicate that InsP3 releases Ca2+ from a pool which is filled up by a Mg2(+) -dependent Ca2+ ATPase.

The important discrepancy between the apparent affinity observed in Ca2+ mobilization studies and the Kd measured in binding studies is a consequence of the quantal process by which inositol 1,4,5-trisphosphate releases Ca2+ from bovine adrenal cortex microsomes.

Inositol 1,4,5-trisphosphate (InsP3) is a second messenger responsible for Ca2+ release from a non-mitochondrial intracellular store. An important discrepancy has been observed between the affinity measured in binding studies (Kd) and the apparent affinity obtained in Ca2+ mobilization studies (EC50). It has been proposed that this discrepancy could be due to different experimental conditions used for Ca2+ mobilization studies and for InsP3 binding studies. With the fluorescent indicator Fura-2, we studied InsP3-induced Ca2+ release activity at 7 degrees C and at 37 degrees C, in bovine adrenal cortex microsomes. Under both conditions, the Ca2+ releasing effect of InsP3 (1 microM) was completed within about 2 s, as a result of the quantal process of InsP3 receptor action. The apparent affinity (EC50) observed for InsP3-induced Ca2+ release at 7 degrees C and at 37 degrees C were 0.64 +/- 0.2 microM and 0.9 +/- 0.2 microM respectively. InsP3 degradation studies, at 37 degrees C, indicated that less than 10% of [3H]-InsP3 was degraded within the first 10 s of incubation. InsP3 association rates were evaluated, at low temperature, with increasing concentrations of [3H]-InsP3. These kinetic studies revealed a direct relationship between the initial rate of association (Vi) and InsP3 concentration. From this relationship, we evaluated that the concentration of InsP3 needed to occupy half of the binding sites within the first second of incubation was 271 nM. We conclude that the discrepancy between Kd and EC50 is related to a kinetic constraint dictated by the quantal process by which InsP3 releases Ca2+.

Thiol-reactive agents biphasically regulate inositol 1,4,5-trisphosphate binding and Ca(2+) release activities in bovine adrenal cortex microsomes.

Within all endocrine cells, the inositol 1,4,5-trisphosphate (InsP(3)) receptor plays an important role in regulation of the intracellular Ca(2+) concentration. In the present study we showed that a single short-term treatment with either N-ethylmaleimide (known to decrease InsP(3) receptor activity) or thimerosal (known to increase InsP(3) receptor activity) caused time-dependent biphasic effects on the InsP(3) binding activity of bovine adrenal cortex microsomes. The early potentiating effect of thiol-reactive agents translated into a 2-fold increase in binding affinity and Ca(2+) release efficiency. The late dampening effect of thiol-reactive agents translated into a continuous reduction of the maximal binding capacity of the microsomes with a concomitant decrease in Ca(2+) release efficiency. Under these conditions, Western blot analyses demonstrated that the level of InsP(3) receptor protein was not modified. Sequential treatments with thimerosal and the reducing agent dithiothreitol showed that the InsP(3) receptor can readily oscillate between high and low affinity states that are related to its alkylation state. Our results suggest a common mode of action of thiol-reactive agents on the InsP(3) receptor. These results also support the contention that cellular mechanisms of thiol group modification could play important roles in regulation of the intracellular Ca(2+) concentration.

Bovine adrenal glomerulosa cells express such a low level of functional B2 receptors that bradykinin does not significantly increase their aldosterone production.

It was recently demonstrated that bradykinin (BK) stimulates aldosterone secretion in bovine adrenal glomerulosa (BAG) cells. The aim of the present study was to characterize the mechanism of action of BK on these cells. Binding experiments with the radioligand 125I-[Tyr8]BK revealed the presence of a relatively small amount (Bmax = 180 +/- 55 fmol/mg of protein) of high affinity (Kd = 0.65 +/- 0.17 nM) binding sites. BK induced a time- and concentration-dependent increase of [3H]inositol trisphosphate ([3H]IP3) in myo-[3H]inositol-labeled BAG cells. A maximal response was obtained with 10 nM BK and the EC50 value was 1.0 +/- 0.5 nM. 125I-[Tyr8]BK binding and BK-induced IP3 production were inhibited by the selective B2 receptor antagonist Icatibant (1 microM) and unaffected by the selective B1 receptor antagonist [DesArg9, Leu8]BK (1 microM). In fura-2 loaded BAG cells, BK (100 nM) induced a typical biphasic Ca2+ response composed of a rapid and transient increase of intracellular Ca2+ concentration [Ca2+]i which slowly declined to a level that remained above basal level for about 5 min. In the presence of EGTA (2 mM), the rapid and transient calcium increase was unaffected whereas the plateau phase was abolished. Angiotensin II (Ang II, 100 nM) also elicited a typical biphasic response in BAG cells. However the rapid and transient elevation of [Ca2+]i was followed by a sustained plateau phase which remained above the basal level for more than 10 min. Although BAG cells express functional B2 receptors, no secretion of aldosterone was observed after stimulation with 100 nM BK for 120 min. Under the same conditions Ang II increased by about 10-fold the basal level of aldosterone. The lack of effect of BK is probably attributable to its very transient effect on IP3 production. Pretreatment of BAG cells with 100 nM BK for 20 min reduced by 70 +/- 10% their total binding capacity. These results suggest a rapid and very efficient desensitization process. We conclude that BAG cells express functional B2 receptors. The weak production of second messengers and the rapid desensitization process could explain why BK fails to increase aldosterone production in these cells. Since functional B2 receptors are expressed in BAG cells it is likely that under some specific physiological or pathological conditions these receptors may play a significant role in aldosterone secretion. However these conditions remain to be determined.

Interaction of polyanions with the recognition sites for inositol 1,4,5-trisphosphate in the bovine adrenal cortex.

Inositol 1,4,5-trisphosphate (InsP3) serves as a second messenger for Ca2+ mobilization in a wide variety of cells. InsP3 activates a specific receptor/channel located on an internal Ca2+ store. Because heparin has already been shown to block the action of InsP3, we have looked at the influence of other polyanions (dextran sulfate and polyvinyl sulfate) on the action and metabolism of InsP3 in the bovine adrenal cortex. Polyvinyl sulfate blocked InsP3 binding to adrenal cortex microsomes with a half-maximal efficiency of 250 nM. Scatchard analyses revealed that this effect was not competitive. The Ca2+ releasing activity of InsP3 on the same microsomal preparation was monitored with the fluorescent indicator, fura-2. Polyvinyl sulfate blocked this activity with a half-maximal efficiency of 80 nM. The effect of polyvinyl sulfate could not be overcome by supramaximal doses of InsP3, suggesting a non-competitive inhibitory effect. The activity of InsP3 phosphatase from bovine adrenal cortex microsomes was also studied. Polyvinyl sulfate inhibited the activity of the phosphatase with a half-maximal efficiency of 5 microM. Lineweaver-Burk plots revealed that this effect was not competitive. Polyvinyl sulfate was able to inhibit the activity of InsP3 kinase from bovine adrenal cortex cytosol. The half-maximal dose was 15 nM and the Lineweaver-Burk analysis showed that the inhibition was not competitive. The effect of dextran sulfate 5000 (DS-5000) on these activities was also studied. DS-5000 inhibited in a competitive manner the binding of InsP3 to its receptor (IC50 of 34 microM), the release of Ca2+ induced by InsP3 (IC50 of 6.5 microM) and the activity of InsP3 phosphatase (IC50 of 57 microM).(ABSTRACT TRUNCATED AT 250 WORDS)

Interaction of benzene 1,2,4-trisphosphate with inositol 1,4,5-trisphosphate receptor and metabolizing enzymes.

In a wide variety of cells, inositol 1,4,5-trisphosphate (InsP3) is an important second messenger involved in the regulation of intracellular Ca2+ concentration. InsP3 interacts with specific receptors and triggers the release of sequestered Ca2+ from an internal store. We have synthesized a structural analogue of InsP3 by phosphorylation of the free hydroxyl groups of 1,2,4-benzenetriol with dibenzylphosphorochloridate. The product benzene 1,2,4-trisphosphate (BzP3) was shown to interact with InsP3 receptor and InsP3 metabolizing enzymes of bovine adrenal cortex. BzP3 competitively blocked InsP3 binding to adrenal cortex microsomes with a half-maximal efficiency at 34 microM. This affinity was about 10,000 times lower than that of InsP3 for its receptor. The Ca2+ releasing activity of BzP3 on the same microsomal preparation was monitored with the fluorescent indicator fura-2. BzP3 had no agonistic effect on this activity but it was able to inhibit InsP3-induced Ca2+ release in a dose-dependent manner. The activity of InsP3 phosphatase was also studied. BzP3 inhibited the activity of the phosphatase with a half-maximal efficiency of 32 microM. BzP3 was also able to inhibit the activity of the cytosolic InsP3 kinase with a half-maximal efficiency of 6.1 microM. These results show that BzP3 is interacting with the three specific recognition sites for InsP3 in the bovine adrenal cortex. The inhibitory effect of this compound is relatively more potent on the metabolizing enzymes than on the Ca(2+)-mobilizing receptor.

Proximate effects of a child sexual abuse prevention program in elementary school children.

OBJECTIVE: The effects of the sexual child abuse prevention program ESPACE were evaluated by means of a Solomon-type design with first and third grade children. ESPACE is an adaptation of the American Child Assault Prevention Program (CAP). Possible side effects of the program were also examined. METHOD: A total of 133 children (64 first-graders and 69 third-graders) participated in the study. Children completed a knowledge questionnaire and a video vignette measure designed to evaluate preventive skills towards abusive and potentially abusive situations. A follow-up measure (2 months) was administered to verify whether knowledge and skills were maintained. RESULTS: Results indicated that children participating in the prevention program showed greater preventive knowledge and skills relative to children not participating. Follow-up data showed that knowledge gains were maintained while the preventive skill gains may attenuate. However, while global skill scores decreased between post-test and follow-up, children still showed greater preventive skills at follow-up than before the program. In terms of unanticipated side effects, results revealed that almost half of the parents noted positive reactions following children's participation in the ESPACE program. Furthermore, the majority of parents did not identify negative reactions in their children following their participation in the workshop. CONCLUSION: The findings suggest that the Quebec adaptation of the CAP program was effective in training children in abuse prevention concepts and skills.

A study of the prevalence of sexual coercion in adolescent heterosexual dating relationships in a Quebec sample.

Very few studies which have addressed the issue of sexual violence in dating relationships have provided prevalence rates for coercion that is perpetrated and sustained for both sexes during adolescence, while adequately accounting for the totality of unwanted sexual experiences. The present study addresses these issues by examining the rates of perpetrated and sustained sexual coercion among 644 French-speaking adolescents between 15 and 19 years of age with heterosexual dating experience from Quebec (Canada). Chi-square analyses reveal that girls are more often the victims of coercion, while boys more frequently perpetrate it. However, some boys are victims of sexual coercion. The most frequently occurring unwanted sexual experiences are kissing, and petting or fondling. Verbal coercion, consisting of continual arguments and pressure, is the strategy most often used by those who perpetrate unwanted sexual experiences, and is the strategy most frequently reported by those who suffer it. More extreme forms of sexual violence are also reported, but at a lower rate. Such high rates of sexual coercion during adolescence, as observed in the present study within a culture which is increasingly open to issues regarding sexuality, argue strongly for the implementation of prevention and intervention programs within this age group.

A pilot behavioural and biological surveillance survey for HIV and other bloodborne infections among Aboriginal people in Regina, Saskatchewan.

BACKGROUND: Aboriginal people in Canada are disproportionately affected by HIV and other blood-borne infections. A-Track is a national public health surveillance system designed to monitor HIV and related infections, behaviours and socio-demographic factors among Aboriginal populations in Canada. The pilot survey for the A-Track surveillance system, the first of its kind in Canada, was conducted in Regina, Saskatchewan and implemented via a community and public health partnership. OBJECTIVE: To assess the prevalence of HIV, hepatitis C, syphilis and associated risk behaviours and socio-demographic factors among Aboriginal people in Regina, Saskatchewan. This focus of the pilot survey was to provide this surveillance information for public health action and to determine whether this type of public health surveillance activity could be conducted in an urban setting across Canada. METHODS: Survey participants were self-identified Aboriginal people (First Nations, Inuit or Métis) or those who claimed Aboriginal ancestry and between the ages of 16 and 60 years. These individuals were also asked to provide a blood sample for HIV, hepatitis C and syphilis antibody testing. Descriptive analyses were performed with sex-based comparisons. RESULTS: There were 1064 people who participated in the survey. Their average age was 33 years and 51% were male. The majority of participants (93%) lived in urban Regina at the time of the survey. Just over half (53.2%) of all participants had been removed from their families during childhood; 29.9% had lived in a residential or boarding school during childhood; and 57.7% had lived at some point in a correctional facility. Among the 1,045 participants who provided a blood sample of sufficient quantity for testing, 5.2% were HIV seropositive and 55.8% of these were aware of their HIV status. The lifetime exposure to hepatitis C was 41.6%, with significantly higher proportions of males than females testing positive for hepatitis C exposure. Syphilis seroprevalence was very low (<1%). Almost three-quarters (71.5%) of participants reported being tested for HIV at least once in their lifetime and among those ever tested, 67.6% had been tested during the 12 months prior to the interview. CONCLUSION: Aboriginal people are disproportionately affected by the HIV/AIDS epidemic in Canada. The findings from the A-Track pilot survey can be used to inform and evaluate prevention and treatment services for HIV and other related infections among Aboriginal people. Lessons learned from the pilot survey could also be used to guide the possible implementation of A-Track in other urban and/or reserve locations in Canada.

The high affinity state of inositol 1,4,5-trisphosphate receptor is a functional state.

Inositol 1,4,5-trisphosphate (InsP3) is a second messenger responsible for the rapid and discontinuous release of Ca2+ from intracellular stores. In this study, the effects of the sulfhydryl reagent thimerosal were investigated on Ca2+ mobilization and on InsP3 binding. Thimerosal was shown to release Ca2+, in a dose-dependent manner, with an EC50 of 135.8 +/- 5.2 microM, from bovine adrenal cortex microsomes. Thimerosal-induced Ca2+ release was not prevented by heparin (250 micrograms/ml), ruling out a participation of InsP3 receptor in that effect. The slow rate of thimerosal-induced Ca2+ release rather suggested an inhibition of microsomal Ca2+ ATPase. At submaximal concentration, thimerosal (100 microM) was also shown to potentiate the release of Ca2+ induced by InsP3. Dose-response experiments revealed that thimerosal enhanced the apparent affinity of InsP3 by a factor 2.21 +/- 0.28, without modifying the maximal amount of Ca2+ released by InsP3. Thimerosal also enhanced, in a dose-dependent manner, [3H]InsP3 binding to adrenal cortex microsomes (EC50 = 43.3 +/- 7.6 microM). A similar effect was also observed on [3H]InsP3 binding to solubilized receptors, suggesting a direct modification of the receptor protein by thimerosal. The effects of thimerosal on Ca2+ release and [3H]InsP3 binding were abolished in the presence of the reducing agent dithiothreitol (1 mM), suggesting a modification by thimerosal of specific thiol groups on these microsomal proteins. Scatchard analysis revealed that thimerosal (100 microM) increased InsP3 receptor affinity by 1.87 +/- 0.26-fold. Kinetic analysis indicated that this increased affinity was due to an enhancement of InsP3 association rate constant. The concomitant increases of binding affinity and Ca2+ releasing potency suggest that the high affinity state of InsP3 receptor is a functional state.

FK506 blocks intracellular Ca2+ oscillations in bovine adrenal glomerulosa cells.

The inositol 1,4,5-trisphosphate (InsP(3)) receptor is a ligand-gated Ca(2+) channel playing an important role in the control of intracellular Ca(2+). In the study presented here, we demonstrate that angiotensin (AngII), phorbol ester (PMA), and FK506 significantly increase the level of InsP(3) receptor phosphorylation in intact bovine adrenal glomerulosa cells. With a back-phosphorylation approach, we showed that the InsP(3) receptor is a good substrate for protein kinase C (PKC) and that FK506 increases the level of PKC-mediated InsP(3) receptor phosphorylation. With a microsomal preparation from bovine adrenal cortex, we showed that PKC enhances the release of Ca(2+) induced by a submaximal dose of InsP(3). We also showed that FK506 blocks intracellular Ca(2+) oscillations in isolated adrenal glomerulosa cells by progressively increasing the intracellular Ca(2+) concentration to a high plateau level. This effect is consistent with an inhibitory role of FK506 on calcineurin dephosphorylation of the InsP(3) receptor, thus keeping the receptor in a phosphorylated, high-conductance state. Our results provide further evidence for the crucial role of the InsP(3) receptor in the regulation of intracellular Ca(2+) oscillations and show that FK506, by maintaining the phosphorylated state of the InsP(3) receptor, causes important changes in the Ca(2+) oscillatory process.

Different populations of inositol 1,4,5-trisphosphate receptors expressed in the bovine adrenal cortex.

The inositol 1,4,5-trisphosphate (InsP3) receptor forms a tetrameric channel responsible for the release of Ca2+ from intracellular stores. In the present study we showed that the experimental approach used to separate bound and free ligands may discriminate between two populations of InsP3 binding sites in bovine adrenal cortex microsomes. A large population of low affinity sites and a small population of high affinity sites were detected with centrifugation and filtration approaches, respectively. Both populations were found in the supernatant and the cytoskeleton fractions of Triton X-100 solubilized microsomes. After treatment of microsomes with thimerosal, an alkylating reagent known to increase InsP3 receptor affinity, the filtration and the centrifugation approaches yielded identical results. With selective anti-InsP3 receptor antibodies, we showed that types 1, 2 and 3 InsP3 receptors are present in intact microsomes and in the cytoskeleton fraction. Binding studies on immunoprecipitated receptors revealed that anti-type 1 antibody recognizes a large population of low affinity sites whereas anti-type 2 antibody recognizes a small population of high affinity sites. Our results indicate that the three types of InsP3 receptors are expressed at different levels in the bovine adrenal cortex. The presence of different types of InsP3 receptors with different ligand binding affinities and their association with the cytoskeleton offer a convenient way for the cell to simultaneously regulate its intracellular Ca2+ concentration and reorganize the spatial distribution of its Ca2+ stores.

Bidirectional activity of the endoplasmic reticulum Ca(2+)-ATPase of bovine adrenal cortex.

It is generally accepted that the ryanodine receptor and the inositol 1,4,5-trisphosphate receptor play major roles in the complex mechanisms by which agonists increase intracellular Ca2+ concentration. In these mechanisms, the endoplasmic reticulum Ca(2+)-ATPase has been attributed an accessory role of refilling the intracellular Ca2+ store. In the present study, the activity of the microsomal Ca(2+)-ATPase of bovine adrenal cortex was investigated. We show that the Ca(2+)-pumping activity of the Ca(2+)-ATPase is related to the ADP/ATP ratio. Our results also show that a brisk increase of the ADP/ATP ratio upon addition of exogenous ADP triggered a rapid release of Ca2+ from preloaded microsomes. ADP released Ca2+ in a dose-dependent manner with an EC50 of 2.98 +/- 0.78 mM. ADP-induced Ca2+ release was not prevented by heparin, ruling out the participation of the inositol 1,4,5-trisphosphate receptor. ADP-induced Ca2+ release could not be attributed to the mere inhibition of the Ca(2+)-ATPase, since the rate of ADP-induced Ca2+ release was 20 times faster than the rate of Ca2+ release induced by a maximal concentration of thapsigargin (2 microM). ADP-induced Ca2+ release experiments performed in the presence of [32P]PO4 revealed a concomitant production of [32P]ATP. ADP-induced [32P]ATP production was dose-dependent, with an EC50 of 5.50 +/- 0.70 mM. ADP-induced [32P]ATP production was prevented by ionomycin (10 microM) and by high concentrations of extramicrosomal Ca2+. These results demonstrate that the microsomal Ca(2+)-ATPase of adrenal cortex possesses a bidirectional activity that depends on ADP concentrations, the Ca2+ gradient across the microsomal membrane, and probably also ATP concentrations.(ABSTRACT TRUNCATED AT 250 WORDS)

Effect of uncoupling agents on AT1 receptor affinity for antagonist analogs of angiotensin II.

AT1 receptor is responsible for most of the physiological effects of Angiotensin II (Ang II). AT1 receptor belongs to the G-protein-coupled receptor (GPCR) family, and it mediates its actions through the coupling of the Gq/11 protein with phospholipase C beta. Classical pharmacology has used the sensitivity of GPCR ligands to uncoupling agents as a criteria to discriminate agonists (which are sensitive) from antagonists (which are insensitive). In this study, the uncoupling agents GTP gamma S and pentosan sulfate (PS) (a low molecular weight polyanion) were used to further characterize the molecular interactions between Ang II analogs and the AT1 receptor. We show that some Ang II antagonists are sensitive to the conformational change of the AT1 receptor induced by uncoupling agents. These results demonstrate that there is no direct relationship between the intrinsic activity of a ligand and its affinity for different conformations of the AT1 receptor and that the sensitivity of GPCR ligands to uncoupling agents can not be used as a criteria to discriminate agonists from antagonists.

Pain hypersensitivity in patients with functional gastrointestinal disorders: a gastrointestinal-specific defect or a general systemic condition?

Visceral hypersensitivity was shown in patients with functional gastrointestinal disorders (FGID). The mechanisms underlying this sensory dysfunction remain undetermined. The initial hypothesis of a generalized reduction in pain tolerance was rejected by further studies that suggested a normal tolerance to somatic stimuli and led to the generally accepted assumption that pain intolerance is specific and exclusive for visceral stimuli in these patients. We wanted to revisit this theory by examining whether patients with FGID reported perception and tolerance to somatic pain differently from normal subjects and whether the response to somatic pain stimulus was correlated to gastrointestinal symptoms or psychological status or distress. Thirty-three patients with FGID (Rome II criteria)(F/M: 26/7; mean age 48+/-9.9) and 33 normal controls (F/M: 24/9; mean age 44.1+/-6.8) were asked to immerse their nondominant hand into 4 degrees C water for as long as possible (maximum 120 sec). Time before appearance of: (1) discomfort, (2) pain, and (3) withdrawing of the hand were noted. The intensity of pain was rated on a visual analog scale from 0 to 100. Self-report questionnaires were used to assess the severity of gastrointestinal symptoms (St-Luc GI index) and the psychological distress (SCL-90) in the patient group. Data are expressed in seconds as mean +/- SEM. Discomfort sensory thresholds were similar in controls and FGID patients (28+/-3 and 24+/-2, respectively; NS) whereas pain and withdrawing were significantly lower in FGID (41+/-3 and 76+/-6 sec) than in controls (62+/-6 and 102+/-4; P < 0.05). Pain intensity was similar in both groups (64+/-4 vs 67+/-3; NS). Female patients showed lower sensory thresholds than male patients and control females (pain thresholds: 39.8+/-3.4 vs 67.8+/-16.7 and vs 56.8+/-8.7; P < 0.05). Sensory thresholds were not different in subgroups of patients with FGID (irritable bowel syndrome and functional dyspepsia). No correlation was shown between sensory thresholds and gastrointestinal index or SCL 90-test. In conclusion, FGID patients showed a threshold to painful somatic stimulus that was lower than in normal subjects. These findings suggest that patients with FGID may have hyperalgesia and low pain tolerance that is not limited to the viscera, but that is part of a systemic general condition.

Induction of collagen mineralization by a bone sialoprotein--decorin chimeric protein.

The observation that hydroxyapatite (HA) formation from metastable solutions can be induced by nucleating proteins such as bone sialoprotein (BSP) suggests a possible treatment for bone defects. The introduction of a mixture of nucleating protein and type I collagen should result in a defect becoming filled with a mineralized collagenous matrix that is biologically and mechanically compatible and capable of being remodeled. To create a nucleating protein that would interact with collagen fibrils, we combined the putative collagen-binding site of mouse decorin with one of two putative HA-nucleating sites of pig BSP. The resulting chimeric protein induced the formation of HA crystals in a steady-state agarose gel system and bound with high affinity to fibrillar type I collagen. The addition of chimeric protein to collagen gels perfused with low concentrations of calcium and phosphate resulted in the deposition of large, apparently needle-shaped HA crystals on the surface of collagen fibrils. These findings suggest that the BSP-decorin chimeric protein could be capable of inducing the mineralization of collagen in vivo.