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The two methods were valid and generally reliable.

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Background. The autonomic nervous system has recently been suggested as a possible physio-pathological pathway in the relation between stress and coronary heart disease. The aim was to examine work stressors in relation to measures of heart rate variability (HRV). Methods. Results are based on observations in a sample of 653 healthy males aged 40-55 years from the Belgian Physical Fitness Study (1976-'78). Data were collected by means of questionnaires and bio-clinical examinations. An index of work stressors was constructed based on 5 items dealing with job satisfaction, physical working conditions, responsibility, work rhythm and social relations. Data on HRV were collected by means of 24-hour ambulatory ECG recordings on a working day. Both time and frequency domain measures of HRV were calculated. Results. The Work Stressor Index was significantly associated with lower pNN50 (% of differences between adjacent normal RR intervals > 50 ms), lower high frequency power and a higher ratio of low frequency over high frequency power. Similar results were obtained after adjusting for age, language, occupation, smoking, body mass index, total cholesterol, systolic blood pressure, and leisure time physical activity. No significant associations were found with SDNN (the SD of all normal RR intervals) and low frequency power. Conclusion. The perception of work stressors was related to reduced HRV in a sample of 653 healthy males. These results suggest that disturbances of the autonomic nervous system and its parasympathetic component in particular may play a role in the link between work stress and coronary heart disease.

(14,15).The N-terminal part of the IP 3 R contains all the structural determinants responsible for specific and selective binding of its physiological agonist, IP 3 (17-20). We have therefore expressed the N-terminal ligand-binding site (first 581 amino acids) of the mouse IP 3 R-1 in Escherichia coli, using a strategy of growth and expression at low temperatures, as described previously by Yoshikawa et al. (20). This protein contains a previously identified Ca 2ϩ -binding region located between amino acids 304 -450 (21). We now demonstrate that Ca 2ϩ and calmodulin can both inhibit IP 3 binding to this recombinant protein and that these inhibitors act independently and additively. Our data indicate that the N-terminal ligand-binding domain of IP 3 R-1 contains regulatory regions directly interacting with Ca 2ϩ and calmodulin. EXPERIMENTAL PROCEDURES Expression of IP 3 R-1 in Sf9Insect Cells-The full-length neuronal mouse IP 3 R-1 cDNA clone containing the S1 splice domain in p400C1 plasmid vector (22) was kindly provided by Drs. K. Mikoshiba and A. Miyawaki (University of Tokyo, Tokyo, Japan). The 5Ј-untranslated region of the original p400C1 clone was removed by polymerase chain reaction (PCR) by amplification of the 5Ј-terminal part up to the Cel-II restriction site (nucleotide 555), before subcloning the IP 3 R-1 cDNA in the baculovirus (Autographa californica) transfer vector pVL 1393 (Invitrogen). Recombinant virus was produced in Spodoptera frugiperda (Sf9) cells by cotransfection of the pVL 1393 IP 3 R-1 construct and the linearized A. californica nuclear polyhydrosis virus DNA (BaculoGold, Pharmingen). The recombinant viruses were purified by isolating individual plaques of transfected cells. These clonal viral populations were amplified by infecting Sf9 cells. The recombinant protein was harvested

The immunophilin FKBP12 associates with intracellular Ca2+ channels and this interaction can be disrupted by the immunosuppressant FK506. We have investigated the effect of FK506 on Ca2+ release and Ca2+ uptake in permeabilized cell types. Changes in medium free [Ca2+] were detected by the fluorescent Ca2+ indicator fluo‐3 in digitonin‐permeabilized SH‐SY5Y human neuroblastoma cells, DT40 and R23‐11 (i.e. triple inositol 1,4,5‐trisphosphate (IP3) receptor knockout cells) chicken B lymphocytes and differentiated and undifferentiated BC3H1 skeletal muscle cells. 45Ca2+ fluxes were studied in saponin‐permeabilized A7r5 rat smooth muscle cells. Addition of FK506 to permeabilized SH‐SY5Y cells led to a sustained elevation of the medium [Ca2+] corresponding to ∼30% of the Ca2+ ionophore A23187‐induced [Ca2+] rise. This rise in [Ca2+] was not dependent on mitochondrial activity. This FK506‐induced [Ca2+] rise was related to the inhibition of the sarcoplasmic/endoplasmic reticulum Ca2+‐Mg2+‐ATPase (SERCA) Ca2+ pump. Oxalate‐facilitated 45Ca2+ uptake in SH‐SY5Y microsomes was inhibited by FK506 with an IC50 of 19 μm. The inhibition of the SERCA Ca2+ pump was not specific since several macrocyclic lactone compounds (ivermectin > FK506, ascomycin and rapamycin) were able to inhibit Ca2+ uptake activity. FK506 (10 μm) did not affect IP3‐induced Ca2+ release in permeabilized SH‐SY5Y and A7r5 cells, but enhanced caffeine‐induced Ca2+ release via the ryanodine receptor (RyR) in differentiated BC3H1 cells. In conclusion, FK506 inhibited active Ca2+ uptake by the SERCA Ca2+ pump; in addition, FK506 enhanced intracellular Ca2+ release through the RyR, but it had no direct effect on IP3‐induced Ca2+ release.

We investigated the interaction of the 12 kDa FK506-binding protein (FKBP12) with two ryanodine-receptor isoforms (RyR1 and RyR3) and with two myo-inositol 1,4,5-trisphosphate (IP $ ) receptor isoforms (IP $ R1 and IP $ R3). Using glutathione Stransferase (GST)-FKBP12 affinity chromatography, we could efficiently extract RyR1 (42p7 % of the solubilized RyR1) from terminal cisternae of skeletal muscle as well as RyR3 (32p4 % of the solubilized RyR3) from RyR3-overexpressing HEK-293 cells. These interactions were completely abolished by FK506 (20 µM) but were largely unaffected by RyR-channel modulators. In contrast, neither IP $ R1 nor IP $ R3 from various sources, including rabbit cerebellum, A7r5 smooth-muscle cells and IP $ R-overexpressing Sf9 insect cells from Spodoptera frugiperda, were retained on the GST-FKBP12 matrix. Moreover, immunoprecipitation experiments indicated a high-affinity interaction of FKBP12 with RyR1 but not with IP $ R1. In order to determine the FKBP12-binding site, we fragmented both RyR1 and IP $ R1