Validity, responsiveness and feasibility of an Italian version of the Canadian Occupational Performance Measure for patients with ankylosing spondylitis.

OBJECTIVES: The purpose of the present study was to produce an Italian version of the Canadian Occupational Performance Measure (COPM) in a group of patients with Ankylosing Spondylitis (AS) and examine the psychometric properties of this version, evaluating its internal consistency, external validity and reliability. Responsiveness and feasibility were also taken into account. METHODS: The Italian COPM, the Italian version of the COPM, was administered to 30 Caucasian patients affected by AS (24 males, 6 females, median age 48 yrs, range 32-67, median disease duration 14 yrs, range 1-30 yrs) together with the Bath Ankylosing Spondylitis Functional Index (BASFI), the Bath Ankylosing Spondylitis Disease Activity (BASDAI) and the Italian version of the Health Assessment Questionnaire (HAQ). Internal consistency was evaluated with Cronbach's alpha for reliability. Construct validity of the COPM was evaluated by a correlation between the BASFI, BASDAI and HAQ scores. Test-retest reliability was assessed by the Intraclass correlation coefficient. Responsiveness and feasibility were also considered. RESULTS: All patients completed the validation study. The questionnaire was internally consistent (alpha coefficient=0.774). A significant correlation was recorded between the COPM and the BASFI (rho=-0.566, p<0.01), BASDAI (rho=-0.491, p<0.01). Test-retest reliability showed a good correlation coefficient and it was confirmed by Bland-Altman method. CONCLUSIONS: The Italian COPM is a valid and reliable instrument focused to detect change in a client's perception of occupational performance over time, in AS patients. Our results confirm the utility of this questionnaire to measure outcome and in planning treatment intervention for patients with AS.

The radiological assessment of axial involvement in psoriatic arthritis: a validation study of the BASRI total and the modified SASSS scoring methods.

OBJECTIVES: To assess the validity of the BASRI and m-SASSS scores for the radiological axial involvement in psoriatic arthritis (PsA). Secondary end-points were to report on clinical, functional and radiographic characteristics of axial involvement. METHODS: Inclusion criteria were satisfaction of the CASPAR criteria and the presence of clinical, functional and/or radiological axial involvement. Three observers scored the radiographs by BASRI and m-SASSS. The construct validity was assessed by examining the correlation of instruments with patient reported outcomes and anthropometric measures. The reliability and the feasibility of the scores were also considered. RESULTS: Seventy-seven patients were enrolled (58 M, 19 F, mean age 49.4 + or - 10.8 yrs, disease duration 13.9 + or - 7.9 yrs). Both instruments showed some modest but significant correlation with clinical measures. When compared, the BASRI showed a correlation with BASMI (rho=0.47, p<0.001), cervical rotation (rho=-0.49, p<0.001), tragus to wall (rho=0.34, p<0.01) and occiput to wall (rho=0.49, p<0.001), modified Schober test (rho=-0.24, p<0.05) and RLDQ (rho=-0.24, p<0.05). When compared, m-SASSS showed a correlation with BASMI (rho=0.39, p<0.001), cervical rotation (rho=-0.41, p<0.001), tragus to wall (rho=0.31, p<0.01) and occiput to wall (rho=0.42, p<0.001), modified Schober and Schober test (rho=-0.34, p<0.001; rho= -0.32, p<0.01), finger to floor (rho=0.37, p<0.01). No correlation was found with BASFI, BASDAI and HAQ. Test-retest showed a good reliability of the scores. Both were feasible but BASRI was the quickest. CONCLUSION: Our results showed that BASRI and m-SASSS were valid instruments for use in spondylitis associated with psoriatic arthritis. Longitudinal data is required to provide sensitivity to change of the two scores.

Effectiveness of rehabilitation in active ankylosing spondylitis assessed by the ASAS response criteria.

OBJECTIVE: To assess the effectiveness of rehabilitation in a group of patients with active ankylosing spondylitis (AS) by the Assessment in Ankylosing Spondylitis (ASAS) Working Group response criteria. METHODS: Fifty-two active AS patients consecutively admitted to a rehabilitation inpatient clinic were enrolled. Patients underwent a 3-week intensive rehabilitation programme and were then discharged with home exercises. The primary outcome measure was the proportion of patients achieving a response based on ASAS 20 at discharge, and at 6 and 12 weeks after. Secondary outcome measures included an improvement in the Revised Leeds Disability Questionnaire (RLDQ) and function expressed as anthropometric measures. RESULTS: The ASAS 20 was achieved in 46 patients (88.5%) at the end of the rehabilitation, in 31 (59.6%) and in 17 (32.7%) patients at 6 and 12 weeks follow-up, respectively. The percentage of ASAS 20 responders statistically declined over time measured from the end of rehabilitation compared with 6 (P < 0.001) and 12 weeks follow-up (P < 0.001). CONCLUSION: The present study shows the effectiveness of rehabilitation as assessed by the ASAS 20, a validated instrument for treatment response, suggesting its usage in rehabilitation settings. Moreover, the results obtained show that the effectiveness of the intensive inpatient rehabilitation declined over time.

Validity and reliability of an Italian version of the revised Leeds disability questionnaire for patients with ankylosing spondylitis.

OBJECTIVE: The purpose of the present study was to produce an Italian version of the Revised Leeds Disability Questionnaire (LDQ) in a group of patients with ankylosing spondylitis, and to examine the psychometric properties of this version, evaluating its internal consistency, external validity and reliability. METHODS: The LDQ was administered to 60 Caucasian patients affected by ankylosing spondylitis (50 males, 10 females, mean age 46.1 +/- 14.2 yr, range 22-74, median disease duration 4.5 yr, range 1-24) together with the Italian version of the Stanford Health Assessment Questionnaire (HAQ), and anthropometric measurements. Thirty patients completed the questionnaire after a 10-day interval. Internal consistency was evaluated with Cronbach's alpha coefficient of reliability. Construct validity of the LDQ was evaluated using the correlation between the HAQ and anthropometric measurements. Test-retest reliability was assessed with the intraclass correlation coefficient. RESULTS: All patients completed the validation study. The questionnaire was internally consistent (alpha=0.90). A significant correlation was recorded between the LDQ and the HAQ score (rho=0.841, P<0.01) and the anthropometric measurements. Test-retest reliability showed a good correlation coefficient (intraclass correlation=0.97). CONCLUSION: The Italian LDQ is a valid and reliable instrument for detecting and measuring functional disability in patients with ankylosing spondylitis. Our results confirm the utility of this questionnaire as a valid and feasible functional measure for patients with ankylosing spondylitis.

Fibromyalgia in patients with irritable bowel syndrome. An association with the severity of the intestinal disorder.

Fibromyalgia (FM) syndrome and irritable bowel syndrome (IBS) are functional disorders in which altered somatic and or visceral perception thresholds have been found. The aim of this study was to evaluate the prevalence of FM in a group of patients with IBS and the possible association of FM with patterns and severity of the intestinal disorder. One hundred thirty consecutive IBS patients were studied. The IBS was divided into four different patterns according to the predominant bowel symptom and into three levels of severity using a functional severity index. All patients underwent rheumatological evaluation for number of positive tender points, number of tender and swollen joints, markers of inflammation, and presence of headache and weakness. Moreover, patients' assessments of diffuse pain, mood and sleep disturbance, anxiety, and fatigue were also measured on a visual analogue scale. The diagnosis of FM was made based on American College of Rheumatology classification criteria. Nonparametric tests were used for statistical analysis. Fibromyalgia was found in 20% of IBS patients. No statistical association was found between the presence of FM and the type of IBS but a significant association was found between the presence of FM and severity of the intestinal disorder. The presence of FM in IBS patients seems to be associated only with the severity of IBS. This result confirms previous studies on the association between the two syndromes.

Peak exercise cardiac power output; a direct indicator of cardiac function strongly predictive of prognosis in chronic heart failure.

OBJECTIVES: This study assessed the prognostic value of peak cardiac power output, measured non-invasively during maximal cardiopulmonary exercise testing, against other exercise-derived haemodynamic variables in patients with chronic congestive heart failure. METHOD AND RESULTS: Two hundred and nineteen unselected, consecutive patients with congestive heart failure (166 men, mean (+/-SD) age of 56+/-13 years) who underwent maximal symptom limited cardiopulmonary treadmill exercise testing with non-invasive estimation of cardiac output using carbon dioxide re-breathing techniques, were followed-up for a mean period of 4.64 (4.47--4.82, 95% CI) years. Cardiac power output was calculated from the product of cardiac output and mean arterial blood pressure. All cause mortality was 12.3% (27 deaths). Peak and resting cardiac power output, peak mean arterial blood pressure, peak and resting cardiac output and peak VO(2)were all predictive of outcome on univariate analyses. Peak cardiac power output, either entered continuously or categorically with a cut-off value of 1.96 watts, was the only independent predictor of mortality (P=0.0004 for values < or >1.96 watts and P=0.001 for continuous values) using multivariate analysis. A relative risk ratio of 5.08 (1.94-13.3, 95% CI) was obtained for a cardiac power output <1.96 watts. CONCLUSION: Peak cardiac power output is an independent predictor of mortality that can be measured non-invasively using cardiopulmonary exercise testing. It can give further prognostic power to a peak VO(2)in the assessment of patients with congestive heart failure.

Distinct vascular patterns of early synovitis in psoriatic, reactive, and rheumatoid arthritis.

OBJECTIVE: To examine the macroscopic vascular pattern of early synovitis in psoriatic arthritis (PsA), reactive arthritis (ReA), and rheumatoid arthritis (RA) and to assess the reliability of the grading features for synovitis. METHODS: Forty-four patients (14 PsA, 12 ReA, and 18 RA) with knee synovitis who were undergoing arthroscopy were assessed. Video recordings of the examination were scored independently by 3 arthroscopists who were blinded to the patient's identity and clinical details. Features of vascularity, villous formation, pannus, granularity, and capillary hyperemia were recorded and kappa values (-1 or =0.8) for features of vascularity, villous hypertrophy, and pannus. Seventy-three percent of the PsA and ReA patients had predominantly tortuous, bushy vessels; 89% of the RA patients had mainly straight, branching vessels. CONCLUSION: The distinct vascular patterns in PsA and ReA compared with those in RA may reflect different specific vascular factors in the pathogenesis of these arthritides. Vascularity and villous hypertrophy are the most reliable features of synovitis grading.

Myocardial oxygenation in dogs during reactive hyperemia.

The mechanisms of myocardial oxygenation during reactive hyperemia were studied in the beating heart using continuous near infrared (NIR) spectroscopy. In open chest dogs, NIR spectroscopy was used to monitor brief occlusions of the left anterior descending artery. These occlusions produced a precipitous drop in tissue oxygen stores (tHbO2+MbO2), tissue blood volume, and the oxidation level of mitochondrial cytochrome a,a3. Reperfusion produced a rapid increase in the NIR signals to supranormal levels, followed by gradual return to baseline. When the duration of occlusion was increased from 20 to 120 s, an essentially linear increase was produced in the overshoot areas defined by the NIR signals. Near infrared spectroscopy (NIRS) separated reactive hyperemia into two phases according to the tissue level of deoxyhemoglobin and deoxymyoglobin (tHb+Mb): (1) an early phase during which the tHb+Mb level was supranormal, reflecting enhanced O2 extraction; and (2) a late phase during which the tHb+Mb level was below baseline, reflecting decreased O2 extraction and increased tissue O2 availability. During reactive hyperemia, when O2 availability was maximal by NIR spectroscopy, O2 consumption was elevated but submaximal, indicating that MVO2 was not limited by O2 availability. Cytochrome a,a3 oxidation state also was restored fully. Thus, myocardial oxygenation is highly regulated during reactive hyperemia. Cellular O2 supply and mitochondrial oxidation state are restored early during reactive hyperemia by increased O2 delivery, increases in tissue blood volume and enhanced O2 extraction. © 1998 Society of Photo-Optical Instrumentation Engineers.

Predicting adverse outcome with exercise SPECT technetium-99m sestamibi imaging in patients with suspected or known coronary artery disease.

The goal of this study was to determine the ability of exercise single-photon emission computed tomographic (SPECT) technetium-99m (Tc-99m) sestamibi imaging to predict adverse events in a population with a comparable distribution of men (n = 114) and women (n = 115). Consecutive patients referred for evaluation of chest pain syndrome, known coronary artery disease, or residual ischemia after acute myocardial infarction underwent imaging using a single-headed SPECT camera. Clinical readings were reviewed and scored by independent observers as normal or abnormal. Follow-up, defined as time from scanning until an event, late revascularization, or patient response averaged 19.2 +/- 5.2 months and was 90% complete (229 of 255 patients). Cardiac death and nonfatal infarction were corroborated by chart review or physician contact. Patients were excluded from analysis if a revascularization procedure was performed within 1 month of imaging. There were 172 patients with normal scans (67%) and 83 with abnormal scans (33%). Of the patients in whom followup was obtained, 2 of 155 with normal scans (0.8%/year) and 6 of 74 with abnormal scans (5.4%/year) had cardiac events. Statistical analysis using the Kaplan-Meier survival curves suggests a significant difference in event-free survival between normal and abnormal scans. Patients with abnormal scans portended a worse outcome (chi-square = 8.04, p <0.005). Thus, exercise SPECT Tc-99m sestamibi scintigraphy is useful for prognostication in a mixed population of patients with suspected or known coronary artery disease in which women comprised 50% of the patient cohort.

Differential sympathetic neural control of oxygenation in resting and exercising human skeletal muscle.

Metabolic products of skeletal muscle contraction activate metaboreceptor muscle afferents that reflexively increase sympathetic nerve activity (SNA) targeted to both resting and exercising skeletal muscle. To determine effects of the increased sympathetic vasoconstrictor drive on muscle oxygenation, we measured changes in tissue oxygen stores and mitochondrial cytochrome a,a3 redox state in rhythmically contracting human forearm muscles with near infrared spectroscopy while simultaneously measuring muscle SNA with microelectrodes. The major new finding is that the ability of reflex-sympathetic activation to decrease muscle oxygenation is abolished when the muscle is exercised at an intensity > 10% of maximal voluntary contraction (MVC). During high intensity handgrip, (45% MVC), contraction-induced decreases in muscle oxygenation remained stable despite progressive metaboreceptor-mediated reflex increases in SNA. During mild to moderate handgrips (20-33% MVC) that do not evoke reflex-sympathetic activation, experimentally induced increases in muscle SNA had no effect on oxygenation in exercising muscles but produced robust decreases in oxygenation in resting muscles. The latter decreases were evident even during maximal metabolic vasodilation accompanying reactive hyperemia. We conclude that in humans sympathetic neural control of skeletal muscle oxygenation is sensitive to modulation by metabolic events in the contracting muscles. These events are different from those involved in either metaboreceptor muscle afferent activation or reactive hyperemia.

Developmental regulation of cytochrome oxidase subunit VIa isoforms in cardiac and skeletal muscle.

Physiological requirements for mitochondrial respiration change during fetal and postnatal development of cardiac and skeletal muscle, particularly after the abrupt transition from the hypoxic fetal environment to the oxygen-rich milieu of the neonate. This study defines the pattern of expression of nuclear genes encoding the muscle-specific (H) and non-muscle-specific (L) isoforms of cytochrome oxidase (COX) subunit VIa during pre- and postnatal development of striated muscles in the mouse. In the early embryo, COX VIa-L was the predominant isoform expressed in all tissues. COX VIa-H mRNA was detectable as early as day 8 postcoitum (pc) in the heart, but not until gestational day 14 in skeletal myofibers of the tongue, diaphragm, and other skeletal muscles. At late fetal stages up until birth (days 16-18 pc), COX VIa-L and COX VIa-H were both expressed in striated myocytes, although the L form remained the dominant isoform. In postnatal animals, however, expression of COX VIa-H increased whereas COX VIa-L decreased in a reciprocal manner. Activation of the COX VIa-H gene also was observed during differentiation of nurine myogenic cells in culture and was followed by diminished expression of the COX VIa-L isoform in maturing myotubes, as in the intact animal. We conclude that regulation of nuclear genes encoding subunits of COX is a component of the developmental programs that govern cardiac and skeletal muscle differentiation and maturation in the mammalian fetus and neonate. COX VIa-L, the predominant isoform in all fetal tissues, is gradually replaced by the muscle-specific H isoform in both cardiac and skeletal muscles, although this transition is not complete until after birth.

Subcellular partitioning of MRP RNA assessed by ultrastructural and biochemical analysis.

A small RNA encoded within the nucleus is an essential subunit of a RNA processing endonuclease (RNase MRP) hypothesized to generate primers for mitochondrial DNA replication from the heavy strand origin of replication. Controversy has arisen, however, concerning the authenticity of an intramitochondrial pool of MRP RNA, and has called into question the existence of pathways for nucleo-mitochondrial transport of nucleic acids in animal cells. In an effort to resolve this controversy, we combined ultrastructural in situ hybridization and biochemical techniques to assess the subcellular partitioning of MRP RNA. Cryosections of mouse cardiomyocytes were hybridized with biotin-labeled RNA probes complementary to different regions of MRP RNA and varying in length from 115 to 230 nucleotides, followed by immunogold labeling. In addition, we transfected mouse C2C12 myogenic cells with constructs bearing mutated forms of the mouse MRP RNA gene and compared the relative abundance of the resulting transcripts to that of control RNAs within whole cell and mitochondrial fractions. In the former analysis we observed preferential localization of MRP RNA to nucleoli and mitochondria in comparison to the nucleoplasm and cytoplasm. In the latter series of studies we observed that wild-type MRP RNA partitions to the mitochondrial fraction by comparison to other RNA transcripts that are localized to the extramitochondrial cytoplasmic space (28S rRNA) or to the nucleoplasm (U1 snRNA). Deletions within 5' or 3' regions of the MRP RNA gene produced transcripts that remain competent for mitochondrial targeting. In contrast, deletion of the midportion of the coding region (nt 118 to 175) of the MRP RNA gene resulted in transcripts that fail to partition to the mitochondrial fraction. We conclude that an authentic intramitochondrial pool of MRP RNA is present in these actively respiring cells, and that specific structural determinants within the MRP RNA molecule permit it to be partitioned to mitochondria.

Myocardial oxygenation in dogs during partial and complete coronary artery occlusion.

Regional myocardial oxygenation was assessed during partial and complete coronary artery occlusion using near infrared spectroscopy. In eight open-chest dogs, partial occlusions resulting in an approximately 42% decrease in left anterior descending coronary artery (LAD) blood flow produced an approximately 21% decrease in tissue O2 stores (tissue oxyhemoglobin plus oxymyoglobin) and no change in the oxidation level of mitochondrial cytochrome aa3. An approximately 81% reduction in LAD blood flow produced nadir levels of tissue oxyhemoglobin plus oxymyoglobin, maximal levels of deoxyhemoglobin plus deoxymyoglobin, a decline in tissue blood volume, and an approximately 39% decrease in cytochrome aa3 oxidation level. These changes were associated with an approximately 52% decrease from the preischemic baseline in mean transmural myocardial blood flow, measured by radiolabeled microspheres, and an approximately 41% decrease in myocardial O2 consumption. Complete occlusion resulted in further decreases in myocardial blood flow, O2 consumption, tissue blood volume, and cytochrome aa3 oxidation state but also produced increases in tissue O2 stores to above the nadir levels noted during partial occlusion. These results indicate that decreases in O2 delivery during partial coronary occlusion increase O2 extraction to sustain mitochondrial O2 availability, but as little as a 52% reduction in myocardial blood flow produces maximal O2 extraction and depletion of tissue O2 stores. Mitochondrial O2 availability is restricted further during complete occlusion because of limited O2 delivery and, possibly, decreases in tissue blood volume and O2 extraction.

Sequence elements required for transcriptional activity of the human myoglobin promoter in intact myocardium.

To define sequence elements required for myoglobin gene transcription in the intact heart, we examined the expression of a reporter gene under the control of a 380-bp upstream segment (-373 to +7) from the human myoglobin gene in transgenic mouse embryos and after gene transfer into left ventricular myocardium of adult rats. This proximal upstream region was sufficient to direct expression of luciferase selectively in cardiac and skeletal muscle of mouse embryos and to recapitulate the pattern of expression of the endogenous mouse myoglobin gene. This same upstream region was transcriptionally active after injection of plasmid DNA into the left ventricular wall of adult rats. Point mutations within two evolutionarily conserved sequence elements--a cytosine-rich (CCAC-box) motif and an A+T-rich (A/T) motif--severely impaired transcription within the intact heart. Nuclear extracts from neonatal cardiomyocytes contain protein factors that bind to each of these elements in a sequence-specific manner. We conclude that combinatorial interactions between the cognate DNA binding factors that recognize these motifs are necessary for transcriptional activity of the myoglobin upstream region in cardiac muscle.

Gradients of transgene expression directed by the human myoglobin promoter in the developing mouse heart.

Prior studies using transient transfection assays in cultured avian and murine skeletal myotubes indicate that the proximal 2-kb segment of the 5' flanking region of the human myoglobin gene contains transcriptional control elements sufficient to direct muscle-specific and developmentally regulated expression of reporter genes. To examine the function of the human myoglobin gene promoter during development of skeletal and cardiac myocytes in the intact animal, a 2.0-kb myoglobin gene upstream fragment was fused to an Escherichia coli lacZ reporter gene and injected into fertilized mouse oocytes. beta-Galactosidase (beta-gal) activity was detected selectively in cardiac and skeletal myocytes of fetal and adult transgenic mice. A distinctive spatial pattern of myoglobin promoter activity was observed in fetal hearts: beta-gal staining was more pronounced within the left ventricular subendocardium than within the subepicardium and was essentially undetectable in the ventricular trabeculae or atria. Expression of endogenous myoglobin mRNA and protein, assessed by in situ hybridization and immunohistochemistry, demonstrated a similar spatial pattern. In contrast, hearts from adult transgenic mice demonstrated essentially homogeneous expression of beta-gal and of endogenous myoglobin mRNA and protein throughout the myocardium, including the trabeculae and atria. These data indicate that the 2.0-kb upstream region of the human myoglobin gene includes cis-acting regulatory elements sufficient to direct transgene expression during murine cardiac development that is myocyte-specific and responsive to positional cues in a similar manner to the endogenous myoglobin gene.

Dynamic mechanisms of cardiac oxygenation during brief ischemia and reperfusion.

Myocardial oxygenation may be altered markedly by changes in tissue blood flow. During brief ischemia and reperfusion produced by transient occlusion of the left anterior descending artery in 10 open-chest dogs, changes in the oxygenation of tissue hemoglobin (Hb) plus myoglobin (Mb) and the oxidation-reduction (redox) state of mitochondrial cytochrome aa3 were monitored continuously using near-infrared spectroscopy. The nondestructive optical technique indicated that coronary occlusion produced an abrupt drop in tissue oxygen stores (tHb02 + Mb02), tissue blood volume (tBV), and the oxidation level of cytochrome aa3. Changes in the cytochrome oxidation state were related inversely to transmural collateral blood flow within the ischemic region (r = 0.77) measured with radiolabeled microspheres. Furthermore, there was a direct relationship (r = 0.91) between collateral blood flow and the tissue level of desaturated Hb and Mb (tHb + Mb). Reperfusion after 2 min of ischemia led to a synchronous overshoot of baseline in coronary flow and tBV followed by supranormal increases in tHb + Mb02 and the oxidation level of cytochrome aa3. The tHb + Mb level increased transiently during reperfusion. This response correlated inversely with collateral flow during ischemia (r = 0.91). Accordingly, the time required to reach peak tHb + Mb levels was shortest in dogs with high collateral flows (r = 0.75). Thus collateral blood flow partially sustains myocardial oxygenation during coronary artery occlusion and influences tissue reoxygenation early during reperfusion.

Isobutylmethylxanthine stimulates adenylate cyclase by blocking the inhibitory regulatory protein, Gi.

The methylxanthines, such as caffeine and theophylline, are an important and widely used class of drugs, which are believed to mediate many of their physiological effects by increasing intracellular concentrations of cAMP. These agents are known to inhibit phosphodiesterases and to block inhibitory A1 adenosine receptors in a competitive manner. Thus, the methylxanthines may increase cAMP accumulation by slowing its inactivation or by enhancing its production. Using a rat adipocyte membrane model we demonstrate that isobutylmethylxanthine (IBMX) induces a dose-dependent 34% increase in cAMP production above that produced by complete phosphodiesterase inhibition with papaverine. This stimulatory effect is dependent upon the inhibitory guanine nucleotide regulatory protein G1, in that inactivation of Gi by pertussis intoxication ablates IBMX-mediated stimulation of adenylate cyclase activity. Because the Gi-dependent effect of IBMX results in increased cAMP production, the mode of action is likely blockade of Gi activity. Accordingly, the capacity of GTP itself to inhibit adenylate cyclase activity is attenuated by IBMX. In contrast to Gi blockade induced by pertussis toxin, this heretofore unappreciated stimulatory mechanism is completely reversed by inhibitory receptor agonists. This mechanism of action may be responsible for certain physiological effects of methylxanthines, which are not easily explained by phosphodiesterase inhibition or antagonism of A1 adenosine receptors.

The new cardiotonic agent sulmazole is an A1 adenosine receptor antagonist and functionally blocks the inhibitory regulator, Gi.

Although many of the new cardiotonic agents are known to increase cAMP and to inhibit with variable potency a low Km cAMP phosphodiesterase, there is still debate as to the mechanism(s) by which these agents act. In a rat adipocyte membrane model we demonstrate that only approximately 50% of the effect of the new cardiotonic agent sulmazole on cAMP accumulation can be attributed to phosphodiesterase inhibition and that the remaining production of cAMP involves stimulation of adenylate cyclase activity. Two distinct pathways for stimulation of adenylate cyclase are herein reported. Sulmazole, UD-CG 212 CL, enoximone, piroximone, amrinone, and milrinone are all shown to be competitive antagonists of inhibitory A1 adenosine receptors, with EC50 values of 11-909 microM. Elimination of the effects of endogenous adenosine with adenosine deaminase reveals a third distinct mechanism for activation of adenylate cyclase. This mechanism appears to involve Gi, the inhibitory guanine nucleotide-regulatory protein, in that sulmazole attenuates the capacity of GTP to inhibit adenylate cyclase activity, and covalent modification of Gi by pertussis toxin treatment abolishes the capacity of sulmazole to mediate stimulation. Thus, functional blockade of Gi activity is the likely mode of action. Restoration of sulmazole's stimulatory effect on adenylate cyclase activity in pertussis toxin-treated membranes can be accomplished by reconstituting purified preparations of either Gi or mixtures of Gi/Go into treated adipocyte membranes. Of note, this stimulatory effect is completely reversed by inhibitory receptor agonists. Thus, the new cardiotonic agent sulmazole mediates increases in cAMP accumulation by mechanisms other than phosphodiesterase inhibition, including A1 adenosine receptor antagonism and inhibition of Gi function.

Heterologous desensitization of the inhibitory A1 adenosine receptor-adenylate cyclase system in rat adipocytes. Regulation of both Ns and Ni.

Adenosine, acting via A1 adenosine receptors, can inhibit adenylate cyclase activity in adipocytes. To assess the effects of chronic adenosine agonist exposure on the A1 adenosine receptor system of adipocytes, rats were infused with (-)-phenylisopropyladenosine or vehicle for 6 days and membranes were prepared. Basal as well as isoproterenol-, sodium fluoride-, and forskolin-stimulated adenylate cyclase activities were significantly increased (approximately 2-fold) in membranes from treated animals. (-)-Phenylisopropyladenosine-mediated inhibition of forskolin-stimulated adenylate cyclase activity was significantly (p = 0.0001) attenuated in membranes from treated rats (20.1 +/- 2.1% inhibition) versus controls (31.6 +/- 2.3% inhibition). Prostaglandin E1-induced inhibition of forskolin-stimulated adenylate cyclase activity was also attenuated: 11.7 +/- 3.6 versus 23.2 +/- 4.6% (p = 0.001). Using the A1 adenosine receptor agonist radioligand (-)-N6-(3-[125I]iodo-4-hydroxyphenylisopropyl)adenosine, 32% fewer high affinity binding sites were detected in membranes from treated animals (p less than 0.04). Photoaffinity labeling with N6-2-(3-[125I]iodo-4-azidophenyl)ethyladenosine revealed no gross difference in receptor structure. The number of beta-adrenergic receptors as well as the percentage of receptors in the high affinity state as assessed by (-)-3-[125I]iodocyanopindolol binding were the same in both groups. In membranes from treated rats, the amount of [alpha-32P]NAD incorporated by pertussis toxin into the alpha subunit of the inhibitory guanine nucleotide regulatory protein (Ni) was decreased by 37 +/- 11%. Concurrently, the quantity of label incorporated by cholera toxin into the alpha subunit of the stimulatory guanine nucleotide regulatory protein (Ns) was increased by 44 +/- 14% in treated membranes. Finally, the capacity of Ns solubilized from treated membranes to stimulate adenylate cyclase activity when reconstituted into cyc- S49 lymphoma cell membranes was enhanced by approximately 50% compared to control. Thus, heterologous desensitization, manifested by a diminished capacity to inhibit adenylate cyclase and an enhanced responsiveness to stimulatory effectors, can be induced in the A1 adenosine receptor-adenylate cyclase system of adipocytes. A decrease in Ni alpha subunit concomitant with an increase in Ns alpha subunit quantity and activity may represent the biochemical mechanism of desensitization in this system.

The rat testicular A1 adenosine receptor-adenylate cyclase system.

When adenosine interacts with membrane-bound A1 receptors, it is capable of inhibiting the enzyme adenylate cyclase in brain and fat tissue. In this paper we characterize the A1 adenosine receptor-adenylate cyclase system of the rat testes. The agonist radioligand (-)-N-[3-[125I]iodo-4-hydroxyphenyl-(isopropyl)adenosine binds with high affinity (Kd, congruent to 1 nM) in a saturable manner (maximum binding, congruent to 600 fmol/mg protein). The A1 adenosine receptor binding displays the appropriate pharmacology, stereospecificity, and sensitivity to guanine nucleotides. The testicular A1 receptor is also coupled in an inhibitory manner to the enzyme adenylate cyclase, as demonstrated by the ability of N6-R-phenyl-2-propyladenosine to inhibit isoproterenol- and forskolin-stimulated cAMP accumulation. The testicular A1 receptor can be solubilized in high yield and in an active form with the detergent digitonin. The solubilized receptor retains all of the pharmacological properties of the membrane-bound receptor. Although there are many similarities among the A1 receptor from testes, brain, and fat tissue, the testicular A1 receptor displays a larger apparent mol wt. By photoaffinity labeling, the A1 adenosine receptor-binding subunit of fat and brain are 38,000 mol wt proteins, while the testicular A1 receptor binding subunit is a 42,000 mol wt protein.