Danuta Stryjek-Kaminska

Photoallergic skin reaction to ribavirin

A 65-yr-old woman with chronic hepatitis C was treated with three million units interferon-αt.i.w. and 1000 mg ribavirin daily. At wk 16 of combination therapy the patient developed an itchy eczematous erythema, partly of urticarial character, which was almost confined to ultraviolet (UV)-exposed sites. Histopathological examination of the skin lesions was consistent with a photoallergic reaction. The minimal erythematous dose for UVA and UVB was assessed on healthy skin. After 24 h, a distinct erythema at the UVB irradiated site was found, whereas no reaction was seen with UVA provocation up to a dose of 10 J/cm2. Correspondingly, determination of the absorption spectrum of ribavirin revealed maximum absorption within UVB at 282.5 nm. Ribavirin was stopped, and the cutaneous lesions and pruritus completely disappeared without subsequent hyperpigmentation. This case indicates that ribavirin is a potential photosensitizer for UVB, which may become increasingly relevant in patients with chronic hepatitis C undergoing combination therapy for 6–12 months with interferon-α and ribavirin.

Epidermal Growth Factor Receptor Signaling in Rat Pancreatic Acinar Cells

Epidermal growth factor (EGF) regulates pancreatic acinar enzyme secretion. The mechanism of action of EGF in pancreatic acinar cells is not clear. In the present study we investigated the role of heterotrimeric GTP-binding proteins (G proteins) in EGF receptor signal transduction. Pancreatic acini were isolated from rat pancreas by collagenase digestion and permeabilized by digitonin. Activation of phosphatidylinositol 4,5-bisphosphate-specific phospholipase C (PLC) was assessed using a radioreceptor assay specific for inositol 1,4,5-trisphosphate [IP3(1,4,5)]. For measurement of amylase secretion isolated pancreatic acini were incubated with secretagogues for 30 min at 37°C. Amylase released into the medium was assessed by monitoring the hydrolysis rate of p-nitrophenyl-α,D-maltoheptaoside. The weakly hydrolyzable GTP analogue guanosine 5′-[3-O-thio]triphosphate (GTPγS) and guanosine 5′-diphosphate (GDP) were used to activate and inhibit G protein-mediated signal transduction, respectively. EGF (90 nM) stimulated amylase release in isolated pancreatic acini. This effect was enhanced by guanosine 5′-[3-O-thio]triphosphate (0.1 mM), which stimulates G proteins. Guanosine 5′-diphosphate (1 mM), which inhibits the activity of heterotrimeric G proteins, had no effect on basal and EGF-induced amylase release. Lower EGF concentrations (20 nM) inhibited COOH-terminal cholecystokinin octapeptide (CCK8)-induced IP3(1,4,5) production and amylase release in pancreatic acini. However, in the presence of GDP, EGF had no significant effect on CCK8-stimulated amylase release. Furthermore, coincubation of the acini with CCK8, EGF, and GDP revealed that GDP reduces the inhibitory effect of EGF on CCK8-induced IP3(1,4,5) production. IP3(1,4,5) accumulation in response to GTPγS was unaffected by EGF. These results suggest a dual mode of action of EGF in pancreatic acini: (1) stimulation of phosphoinositide-specific, PLC-mediated cellular responses at high EGF concentrations by a G protein-insensitive mechanism and (2) inhibition of PLC at low EGF concentrations, which might involve either activation of inhibitory G proteins or uncoupling of the CCK receptor from activation of PLC.

Epidermal growth factor inhibits hormone- and fibroblast growth factor-induced activation of phospholipase C in rat pancreatic acini.

Epidermal growth factor (EGF) inhibits cholecystokinin-octapeptide-stimulated amylase release and inositol 1,4,5-trisphosphate (1,4,5-IP3) production in isolated rat pancreatic acini. In the present study, pancreatic acini were used to investigate the effect of EGF on amylase release and 1,4,5-IP3 production induced by secretagogues that activate either phospholipase C-beta (carbachol, bombesin) or phospholipase C-gamma [basic fibroblast growth factor (bFGF)]. The results show that EGF (100 ng/ml) inhibited bombesin (0.1 nM-1 microM)-induced amylase release almost completely. Similarly, the effect of EGF on carbachol-stimulated amylase release was substantial at submaximal (0.1 microM: 44% inhibition), maximal (1 microM: 75% inhibition), and supramaximal (100 microM: 33% inhibition) carbachol concentrations. EGF reduced amylase release at submaximal bFGF concentrations (0.1 nM: 40% inhibition), but not at supramaximal bFGF concentrations (1 and 10 nM). EGF decreased the peak increase of 1,4,5-IP3 in response to bombesin and carbachol (5 s after beginning of the incubation) and bFGF (15 s after beginning of the incubation) by 81 +/- 19%, 65 +/- 15%, and 56 +/- 18%, respectively. Receptor binding characteristics for secretagogues that activate phospholipase C were not influenced by coincubation with EGF excluding heterologous transmembrane receptor modulation. These results suggest that EGF inhibits the action of phospholipase C-beta- and gamma-isoenzyme-activating secretagogues in the exocrine pancreas by a postreceptor mechanism.

Pertussis toxin‐sensitive G‐proteins inhibit fibroblast growth factor‐induced signaling in pancreatic acini

Signal transduction of fibroblast growth factor (FGF) receptors is known to involve tyrosine phosphorylation of several substrates, including Grb2, phospholipase C‐γ, and phosphatidylinositol 3‐kinase, whereas the role of G‐proteins in FGF receptor signaling is controversial. In the present study we investigated the role of G‐proteins in FGF receptor signaling in rat pancreatic acini. Immunological analysis revealed the presence of FGF receptor and phospholipase C‐γ1 in rat pancreatic acini. Both basic fibroblast growth factor (FGF‐2) and guanosine 5′‐(γ‐O‐thio)triphosphate (GTPγS) caused an increase in inositol 1,4,5‐trisphosphate (1,4,5‐IP3) production and amylase release. Combined stimulation of the acini with GTPγS and FGF‐2 led to a decrease of these responses as compared to the effect of the single substances. When pancreatic acini were preincubated with FGF‐2 (1 nM) or vehicle (water) ADP‐ribosylation of the α‐subunit of Gi‐type G‐proteins by pertussis toxin was reduced in membranes prepared from FGF‐2 pretreated acini as compared to control acini, suggesting functional interaction of FGF receptors with Gi‐proteins. Pretreatment of acini with pertussis toxin which inhibits Gi‐type G‐proteins abolished the inhibitory effect of GTPγS on FGF‐induced 1,4,5‐IP3 production and amylase release, whereas the stimulatory effects of FGF‐2 and GTPγS on these parameters remained unchanged. In conclusion, these results show communication of FGF receptors and Gi‐type G‐proteins and that Gi‐type G‐proteins exert an inhibitory influence on FGF‐induced activation of phosphoinositide‐specific phospholipase C in pancreatic acinar cells.

Effects of guanine nucleotides on bombesin-stimulated signal transduction in rat pancreatic acinar cells

To study the role of guanine nucleotide binding proteins (G proteins) in bombesin receptor signal transduction, we investigated the effects of guenine nucleotide analogues and of the G protein activator NaF on bombesin-induced amylase release, inositol 1,4,5-trisphosphate (Ins(1,4,5)P3) production and release of intracellular Ca2+ in rat pancreatic acini. In digitonin-permeabilized acini, guanosine 5′-[γ-thio]triphosphate (GTPγS), a well-known activator of G proteins, potentiated bombesin-induced Ins(1,4,5)P3 production and increased amylase release at low bombesin concentrations (<10 nM). By contrast, GTPγS decreased bombesin-stimulated amylase release at high bombesin concentrations (>10 nM). Fluoride (10 mM), another G protein activator, had similar effects to GTPγS on amylase release. However, unlike GTPγS it had no effect on Ins(1,4,5)P3 production and release of intracellular Ca2+ induced by high bombesin concentrations. GDP and its analogues, such as 2′-desoxyguanosine 5′-diphosphate (dGDP) or guanosine 5′-[β-thio]diphosphate (GDPβS), inhibit activation of G proteins. GDP and dGDP both inhibited amylase release and Ins(1,4,5)P3 production at all bombesin concentrations tested. In contrast, GDPβS mimicked the effects of GTPγS on bombesin-stimulated amylase release and Ins(1,4,5)P3 accumulation. In conclusion, we suggest that bombesin receptor-mediated signal transduction involves G proteins in pancreatic acini. The correlation between inhibition of maximum-stimulated enzyme secretion and further increase in Ins(1,4,5)P3 production in response to GTPγS at high bombesin concentrations suggests that overstimulation of phospholipase C inhibits amylase release. The discrepant effects of GDP and of GDPβS on phospholipase C activity and amylase release might be due to the ability of GDPβS, but not of GDP to activate G proteins persistently after phosphorylation by G protein-associated GDP kinases.