Development of a recombinant cell-based system for the characterisation of phosphodiesterase 4 isoforms and evaluation of inhibitors.

We described the development of a recombinant cell-based system for the characterisation of phosphodiesterase (PDE) 4 isoforms and the evaluation of inhibitors. The Chinese hamster ovary (CHO) cell, which was found to have a low endogenous PDE4 background and no beta-adrenergic receptors (beta-AR), was transiently transfected with beta-AR and various PDE4 isoforms which were expressed as functionally coupled molecules. From correlations of elevation of adenosine 3',5'-cyclic monophosphate in situ and the inhibition of catalytic activity in vitro with the various PDE4 isoforms, it was apparent that PDE4A4, 4B2, 4C2, 4D2, and 4D3 all adopted a high-affinity binding conformation (i.e. expressed the high-affinity rolipram binding site) in the CHO cell, whereas PDE4A330 was expressed in a low-affinity conformation in situ. This gives the opportunity of using this system to screen and optimise inhibitors against a low-affinity conformation of PDE4 in situ and use a high-affinity conformation of PDE4 as a counterscreen, as inhibitor activity against this conformer has been linked with undesirable side effects. This system could also be utilised to screen inhibitors against various PDE4 isoforms in isolation against a low endogenous PDE background in situ for isoform-selective inhibitors.

Chromium improves insulin response to glucose in rats.

The effects of chromium (Cr) supplementation on insulin secretion and glucose clearance (KG) during intravenous glucose tolerance tests (IVGTTS) were assessed in rats with impaired glucose tolerance due to dietary Cr deficiency. Male Wistar rats were maintained after weaning on a basal low-Cr diet containing 55% sucrose, 15% lard, 25% casein. American Institute of Nutrition (AIN)-recommended levels of vitamins, no added Cr, and an altered mineral content as required to produce Cr deficiency and impaired glucose tolerance. The Cr-supplemented group ([+Cr] n = 6) were provided with 5 ppm Cr as CrCl3 in the drinking water, and the Cr-deficient group ([-Cr]n = 5) received purified drinking water. At 12 weeks on the diet, both groups of rats were hyperinsulinemic (+Cr, 103 +/- 13; -Cr, 59 +/- 12 microU/mL) and normoglycemic (+Cr, 127 +/- 7; -Cr, 130 +/- 4 mg/dL), indicating insulin resistance. After 24 weeks, insulin levels were normal (+Cr, 19 +/- 5; -Cr, 21 +/- 3 microU/mL) and all rats remained normoglycemic (+Cr, 124 +/- 8; -Cr, 131 +/- 6 mg/dL). KG values during IVGTTS were lower in -Cr rats (KG = 3.58%/min) than in +Cr rats (KG = 5.29%/min), correlating with significantly greater 40-minute glucose areas in the -Cr group (P < .01). Comparisons of 40-minute insulin areas indicated marked insulin hyperresponsiveness in the -Cr group, with insulin-secretory responses increased nearly twofold in -Cr animals (P < .05). Chromium deficiency also led to significant decreases in cyclic adenosine monophosphate (cAMP)-dependent phosphodiesterase (PDE) activity in spleen and testis (P < .01). In these studies, Cr deficiency was characterized by both beta-cell hypersecretion of insulin and tissue insulin resistance that were associated with decreased tissue levels of cAMP PDE activity.

Cyclic AMP-reactive proteins in human saliva.

This study was conducted to compare cyclic AMP-reactive proteins (cARP), the secretory form of regulatory (R) subunits of cyclic AMP-dependent protein kinase (PKA), and cyclic nucleotide phosphodiesterase (PDE) activity in human whole saliva with that of parotid fluid. Additionally, experiments were done to determine whether secretory cARP is altered by environmental stimuli. Earlier work showed that R subunits are present in parotid fluid and in salivary glands of rats. No previous information is available about secretory PDE in saliva. Whole and parotid ductal saliva samples were collected by a non-invasive procedure from healthy volunteers. After photoaffinity labelling with [32P]-8-N3-cAMP, the R subunits were identified by autoradiography. Cyclic nucleotide PDE activity was measured as a function of the conversion of the cyclic nucleotide to the tritiated 5'-nucleotide. The results showed that R of the type II cAPK, RII (M(r) 50-54 kDa) and/or a slower-moving isoform (M(r) 54-56 kDa, RIIa) were present in all parotid saliva samples tested. Whole saliva was positive for RII in more than 95% of the samples tested (n = 62), but with 50-90% reduction in concentration compared to parotid fluid. Both female and male subjects exposed to controlled auditory (60-80 dB) stimuli responded by a two- to five-fold increase in photoaffinity labelling of cARP (salivary RII, RIIa and RIIfr). There was considerable individual variability, but in all cases the differences in the results were significant (p < 0.05, n = 20). Whole saliva showed measurable PDE activity in fresh or frozen samples, whereas no PDE activity was detected in parotid fluid.(ABSTRACT TRUNCATED AT 250 WORDS)

Croton oil- and benzo(a)pyrene-induced changes in cyclic adenosine 3':5'-monophosphate and cyclic guanosine 3':5'-monophosphate phosphodiesterase activities in mouse epidermis.

The topical application of croton oil, benzo(a)pyrene, acetic acid, and 12-O-tetradecanoyl-phorbol-13-acetate to mouse skin caused an increase in the activity of epidermal low-affinity cyclic adenosine 3':5'-monophosphate (cyclic AMP) phosphodiesterase. The increase was most pronounced with croton oil, began between 4 and 6 hr after application of this material, and was maintained for at least 48 hr. The activity of cyclic guanosine 3':5'-monophosphate phosphodiesterase was also increased by treatment with croton oil or 12-O-tetradecanoyl-phorbol-13-acetate, but detailed time courses were not obtained. Increased activity was observed in both the soluble fractions and the washed particulate fractions of epidermis. Fractionation of soluble extracts from acetone-treated epidermis on DEAE-cellulose columns showed the presence of enzymes with specificity for both cyclic AMP and cyclic guanosine 3':5'-monophosphate, together with a peak catalyzing the hydrolysis of both cyclic AMP and cyclic guanosine 3':5'-monophosphate. The activity of this latter nonspecific activity was selectively increased following treatment with croton oil. The increase in cyclic AMP phosphodiesterase activity was partially abolished by multiple injections of cycloheximide, suggesting that new protein synthesis was involved. Injection of the alpha-receptor antagonist phentolamine abolished a croton oil-induced rise in epidermal cyclic AMP levels and decreased the induction of cyclic AMP phosphodiesterase activity. From these results it was concluded that the increase in enzyme activity was induced by cyclic AMP.