Smooth muscle contraction is activated primarily by the Ca2+‐calmodulin (CaM)‐dependent phosphorylation of the 20 kDa light chains (LC20) of myosin. Activation can also occur in some instances without a change in intracellular free [Ca2+] or indeed in a Ca2+‐independent manner. These signalling pathways often involve inhibition of myosin light chain phosphatase and unmasking of basal kinase activity leading to LC20 phosphorylation and contraction. We have used demembranated rat caudal arterial smooth muscle strips and isolated chicken gizzard myofilaments in conjunction with the phosphatase inhibitor microcystin‐LR to investigate the mechanism of Ca2+‐independent phosphorylation of LC20 and contraction. Treatment of Triton X‐100‐demembranated rat caudal arterial smooth muscle strips with microcystin at pCa 9 triggered a concentration‐dependent contraction that was slower than that induced by pCa 4.5 or 6 but reached comparable steady‐state levels of tension. This Ca2+‐independent, microcystin‐induced contraction correlated with phosphorylation of LC20 at serine‐19 and threonine‐18. Whereas Ca2+‐dependent LC20 phosphorylation and contraction were inhibited by a synthetic peptide (AV25) based on the autoinhibitory domain of myosin light chain kinase (MLCK), Ca2+‐independent, microcystin‐induced LC20 phosphorylation and contraction were resistant to AV25. Ca2+‐independent LC20 kinase activity was also detected in chicken gizzard smooth muscle myofilaments and catalysed phosphorylation of endogenous myosin LC20 at serine‐19 and/or threonine‐18. This is in contrast to MLCK which phosphorylates threonine‐18 only after prior phosphorylation of serine‐19. Gizzard Ca2+‐independent LC20 kinase could be separated from MLCK by differential extraction from myofilaments and by CaM affinity chromatography. Its activity was resistant to AV25. We conclude that inhibition of smooth muscle myosin light chain phosphatase (MLCP) unmasks the activity of a Ca2+‐independent LC20 kinase associated with the myofilaments and distinct from MLCK. This kinase, therefore, probably plays a role in Ca2+ sensitization and Ca2+‐independent contraction of smooth muscle in response to stimuli that act via Ca2+‐independent pathways, leading to inhibition of MLCP.

N-(1,3-Dimethylbutyl)-N′-phenyl-p-phenylenediamine-quinone (6PPD-quinone), a transformation product of the rubber tire antioxidant 6PPD, has recently been identified as the chemical responsible for urban runoff mortality syndrome in coho salmon, with a median lethal concentration (LC50) of <0.1 μg/L. Subsequent studies have failed to confirm comparable sensitivity in other fish species. Here, we investigated the acute toxicity of 6PPD-quinone to rainbow trout, brook trout, Arctic char, and white sturgeon. Fish were exposed under static renewal conditions, and exposure concentrations were verified analytically. Mortalities in brook trout occurred between 1.2 and 20 h, while mortalities began after 7 h and spanned 60 h in rainbow trout. The LC50s in brook trout (24 h) and rainbow trout (72 h) were 0.59 and 1.00 μg/L, respectively. Both species showed characteristic symptoms (increased ventilation, gasping, spiraling, and loss of equilibrium) shortly before death. No mortalities were observed after exposure of either char or sturgeon for 96 h at measured concentrations as high as 14.2 μg/L. This is the first study to demonstrate the acute toxicity of 6PPD-quinone to other fishes of commercial, cultural, and ecological importance at environmentally relevant concentrations and provides urgently needed information for environmental risk assessments of this contaminant of emerging concern.

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The investigation established and validated methods to measure multiple biochemical indices of condition (whole body total lipids, whole body triglycerides, muscle RNA : DNA ratio and muscle protein) simultaneously in the same individual juvenile fish. It also provided examples of their application using a species comparison (salmonids and cyprinids) of the degree of change in these indices after food deprivation. The results showed that juvenile rainbow trout Oncorhynchus mykiss were much more susceptible to a decline in all biochemical indices of condition than juvenile fathead minnows Pimephales promelas upon food deprivation. The combination of these techniques can be used to accurately assess condition and growth potential of individuals from wild fish populations.