Procedural pain does not raise plasma levels of cortisol or catecholamines in adult intensive care patients after cardiac surgery.

The gold standard for quantification of pain is a person's self-report. However, we need objective parameters for pain measurement when intensive care patients, for example, are not able to report pain themselves. An increase in pain is currently thought to coincide with an increase in stress hormones. This observational study investigated whether procedure-related pain is associated with an increase of plasma cortisol, adrenaline, and noradrenaline. In 59 patients receiving intensive care after cardiac surgery, cortisol, adrenaline, and noradrenaline plasma levels were measured immediately before and immediately after patients were turned for washing, either combined with the removal of chest tubes or not. Numeric rating scale scores were obtained before, during, and after the procedure. Unacceptably severe pain (numeric rating scale ≥ 4) was reported by seven (12%), 26 (44%), and nine (15%) patients, before, during and after the procedure, respectively. There was no statistically significant association between numeric rating scale scores and change in cortisol, adrenaline, and noradrenaline plasma levels during the procedure. Despite current convictions that pain coincides with an increase in stress hormones, procedural pain was not associated with a significant increase in plasma stress hormone levels in patients who had undergone cardiac surgery. Thus, plasma levels of cortisol, adrenaline, and noradrenaline seem unsuitable for further research on the measurement of procedural pain.

Fingerprinting of adenylyl cyclase activities during Dictyostelium development indicates a dominant role for adenylyl cyclase B in terminal differentiation.

Activation of cAMP-dependent protein kinase (PKA) triggers terminal differentiation in Dictyostelium, without an obvious requirement for the G-protein-coupled adenylyl cyclase, ACA, or the osmosensory adenylyl cyclase, ACG. A third adenylyl cyclase, ACB, was recently detected in rapidly developing mutants. The specific characteristics of ACA, ACG, and ACB were used to determine their respective activities during development of wild-type cells. ACA was highly active during aggregation, with negligible activity in the slug stage. ACG activity was not present at significant levels until mature spores had formed. ACB activity increased strongly after slugs had formed with optimal activity at early fruiting body formation. The same high activity was observed in slugs of ACG null mutants and ACA null mutants that overexpress PKA (acaA/PKA), indicating that it was not due to either ACA or ACG. The detection of high adenylyl cyclase activity in acaA/PKA null mutants contradicts earlier conclusions (B. Wang and A. Kuspa, Science 277, 251-254, 1997) that these mutants can develop into fruiting bodies in the complete absence of cAMP. In contrast to slugs of null mutants for the intracellular cAMP-phosphodiesterase REGA, where both intact cells and lysates show ACB activity, wild-type slugs only show activity in lysates. This indicates that cAMP accumulation by ACB in living cells is controlled by REGA. Both REGA inhibition and PKA overexpression cause precocious terminal differentiation. The developmental regulation of ACB and its relationship to REGA suggest that ACB activates PKA and induces terminal differentiation.

Mutation of an EF-hand Ca(2+)-binding motif in phospholipase C of Dictyostelium discoideum: inhibition of activity but no effect on Ca(2+)-dependence.

Phosphoinositide-specific phospholipase C (PLC) is dependent on Ca2+ ions for substrate hydrolysis. The role of an EF-hand Ca(2+)-binding motif in Ca(2+)-dependent PLC activity was investigated by site-directed mutagenesis of the Dictyostelium discoideum PLC enzyme. Amino acid residues with oxygen-containing side chains at co-ordinates x, y, z, -x and -z of the putative Ca(2+)-binding-loop sequence were replaced by isoleucine (x), valine (y) or alanine (z, -x and -z). The mutated proteins were expressed in a Dictyostelium cell line with a disrupted plc gene displaying no endogenous PLC activity, and PLC activity was measured in cell lysates at different Ca2+ concentrations. Replacement of aspartate at position x, which is considered to play an essential role in Ca2+ binding, had little effect on Ca2+ affinity and maximal enzyme activity. A mutant with substitutions at both aspartate residues in position x and y also showed no decrease in Ca2+ affinity, whereas the maximal PLC activity was reduced by 60%. Introduction of additional mutations in the EF-hand revealed that the Ca2+ concentration giving half-maximal activity was unaltered, but PLC activity levels at saturating Ca2+ concentrations were markedly decreased. The results demonstrate that, although the EF-hand domain is required for enzyme activity, it is not the site that regulates the Ca(2+)-dependence of the PLC reaction.